Methods for treating progressive multiple sclerosis

ABSTRACT

The present invention concerns methods for treating progressive multiple sclerosis (MS) in a patient, and an article of manufacture with instructions for such use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit to U.S. Provisional ApplicationSer. No. 61/097,464, filed Sep. 16, 2008, the entirety of which ishereby incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns methods for treating progressive multiplesclerosis (MS) in a patient, and an article of manufacture withinstructions for such use.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392006102 SEQLIST.TXT,date recorded: Jul. 13, 2015, size: 38 KB).

BACKGROUND OF THE INVENTION Multiple Sclerosis

Multiple Sclerosis (MS) is an inflammatory and demyelinatingdegenerative disease of the human central nervous system (CNS). It is aworldwide disease that affects approximately 300,000 persons in theUnited States; it is a disease of young adults, with 70%-80% havingonset between 20 and 40 years old (Anderson et al. Ann Neurology31(3):333-6 (1992); Noonan et al. Neurology 58:136-8 (2002)). MS is aheterogeneous disorder based on clinical course, magnetic resonanceimaging (MRI) scan assessment, and pathology analysis of biopsy andautopsy material (Lucchinetti et al. Ann Neurol 47:707-17 (2000)). Thedisease manifests itself in a large number of possible combinations ofdeficits, including spinal cord, brainstem, cranial nerve, cerebellar,cerebral, and cognitive syndromes. Progressive disability is the fate ofmost patients with MS, especially when a 25-year perspective isincluded. Half of MS patients require a cane to walk within 15 years ofdisease onset. MS is a major cause of neurologic disability in young andmiddle-aged adults and, until the past decade, has had no knownbeneficial treatments. MS is difficult to diagnose because of thenon-specific clinical findings, which led to the development of highlystructured diagnostic criteria that include several technologicaladvances, consisting of MRI scans, evoked potentials, and cerebrospinalfluid (CSF) studies. All diagnostic criteria rely upon the generalprinciples of scattered lesions in the central white matter occurring atdifferent times and not explained by other etiologies such as infection,vascular disorder, or autoimmune disorder (McDonald et al. Ann Neurol50:121-7 (2001)). MS has four patterns of disease: relapsing-remittingMS (RRMS; 80%-85% of cases at onset), primary progressive MS (PPMS;10%-15% at onset), progressive relapsing MS (PRMS; 5% at onset); andsecondary progressive MS (SPMS) (Kremenchutzky et al. Brain 122 (Pt10):1941-50 (1999); Confavreux et al. N Engl J Med 343(20):1430-8(2000)). An estimated 50% of patients with RRMS will develop SPMS in 10years, and up to 90% of RRMS patients will eventually develop SPMS(Weinshenker et al. Brain 112(Pt 1):133-46 (1989)).

Currently, six drugs in four classes are approved in the United Statesfor the treatment of RRMS, whereas no drugs have been approved for PPMS.The RRMS treatments include the following: interferon class, IFN-beta-la(REBIF® and AVONEX®) and IFN-beta-lb (BETASERON®); glatiramer acetate(COPAXONE®), a polypeptide; natalizumab (TYSABRI®); and mitoxantrone(NOVANTRONE®), a cytotoxic agent. Other drugs have been used withvarying degrees of success, including corticosteroids, methotrexate,cyclophosphamide, azathioprine, and intravenous (IV) immunoglobulin. Thebenefits of currently approved treatments are relatively modest (˜30%)for relapse rate and prevention of disability in RRMS as suggested bytwo meta-analyses (Filippini et at Lancet 361:545-52 (2003)).

Other clinical studies evaluated other immunomodulatory agents in MS,including tumor necrosis factor-α inhibitors and altered peptideligands, which aggravated rather than improved MS (Lenercept MultipleSclerosis Study Group and the University of British Columbia MS/MRINeurology 53:457-65 (1999); Bielekova et al. Nat Med 2000; 6:1167-75(2000), erratum appears in Nat Med 6:1412 (2000)).

The predominant view of MS pathophysiology has held that inflammation isprincipally mediated by CD4⁺ Th1 T cells. Therapeutic approaches basedon this theory such as IFN-beta and glatiramer acetate decrease, but donot fully prevent, occurrence of exacerbations or accumulation ofdisability.

The existence of a humoral component in human MS has been implicitlyrecognized for decades, as evidenced by inclusion of CSF oligoclonalbands and increased intrathecal IgG synthesis in diagnostic criteria forMS (Siden A. J Neurol 221:39-51(1979); McDonald et al. Ann Neurol50:121-7 (2001); Andersson et al Eur J Neurol 9:243-51 (2002); O'Connor,P. Neurology 59:S1-33 (2002)). The presence of oligoclonal bands,increased free light chains, and increased intrathecal IgM synthesiscorrelates with MS disease activity and may be a predictor of moresevere outcomes (Rudick et al. Mult Scler 1:150-5 (1995); Zeman et al.Acta Cytol 45:51-9 (2001); Izquierdo et al. Acta Neurol Scand 105:158-63(2002); Wolinsky J. J Neurol Sci 206:145-52 (2003); Villar et al. AnnNeurol 53:222-6 (2003)).

Anti-myelin antibodies (myelin basic protein (MBP) and myelinoligodendrocyte glycoprotein (MOG)) have been detected in the serum ofpatients with progressive and relapsing forms of MS (Reindl et al. Brain122:2047-56 (1999); Egg et al. Mult Scler 7(5):285-9 (2001)).Anti-myelin antibodies have also been detected in the CSF of MS patients(Reindl et al. Brain 122:2047-56 (1999); Egg et al. Mult Scler7(5):285-9 (2001); Andersson et al. Eur J Neurol 9:243-51 (2002)).Additional types of antibodies such as anti-ganglioside antibodies oranti-neurofilament antibodies have been observed in patients with MS(Mata et al. Mult Scler 5:379-88 (1999); Sadatipour et al. Ann Neurol44:980-3 (1998)). A report indicated that the presence of serum anti-MOGand anti-MBP antibodies was a strong predictor of progression from aclinically isolated demyelinating event to definite RRMS (Berger et al.N Engl J Med 349:139-45 (2003)). The adjusted hazard ratio forexperiencing an exacerbation was 76.5 for patients who were seropositivefor both antibodies and 31.6 for patients who were seropositive only foranti-MOG.

An international pathology consortium found that antibodies bound tomyelin are present in the majority of patients with MS, with plasmacells and B cells also found in MS lesions, providing additionalevidence for a humoral role in MS (Prineas and Wright, Lab Invest38:409-21 (1978); Esiri M. Neuropathol Appl Neurobiol 6:9-21 (1980);Genain et al. Nat Med 5:170-5 (1999); Lucchinetti et al. Ann Neurol47:707-17 (2000); Wingerchuk et al. Lab Invest 81:263-81 (2001)). Bcells are detectable in the CSF of patients with MS, and the presence ofa relatively high proportion of B cells may be predictive of more severedisability progression (Cepok et al. Brain 124(Pt 11):2169-76 (2001)).

In patients with RRMS or opsoclonus-myoclonus syndrome, Rituximabreportedly depleted peripheral B-cells in all patients and decreased thenumber of CSF B cells in some patients (Pranzatelli et al. Neurology60(Suppl1) P05.128:A395 (2003); Cross et al. “Preliminary Results from aPhase II Trial of Rituximab in MS” (abstract) Eighth Annual Meeting ofthe Americas Committees for Research and Treatment in Multiple SclerosisACTRIMS 20-1 (October, 2003); Cross et al. J Neuroimmunol. 180:63-70(2006)). See also Cree et al. “Tolerability and Effects of Rituximab“Anti-CD20 Antibody” in Neuromyelitis Optica and Rapidly WorseningMultiple Sclerosis” Meeting of the Am. Acad. Neurol. (April, 2004); Creeet al. Neurology 64:1270-2 (2005).

CD20 Antibodies and Therapy Therewith

Lymphocytes are one of many types of white blood cells produced in thebone marrow during the process of hematopoiesis. There are two majorpopulations of lymphocytes: B lymphocytes (B cells) and T lymphocytes (Tcells). The lymphocytes of particular interest herein are B cells.

B cells mature within the bone marrow and leave the marrow expressing anantigen-binding antibody on their cell surface. When a naive B cellfirst encounters the antigen for which its membrane-bound antibody isspecific, the cell begins to divide rapidly and its progenydifferentiate into memory B cells and effector cells called “plasmacells”. Memory B cells have a longer life span and continue to expressmembrane-bound antibody with the same specificity as the original parentcell. Plasma cells do not produce membrane-bound antibody but insteadproduce the antibody in a form that can be secreted. Secreted antibodiesare the major effector molecule of humoral immunity.

The CD20 antigen (also called human B-lymphocyte-restricteddifferentiation antigen, Bp35) is a hydrophobic transmembrane proteinwith a molecular weight of approximately 35 kD located on pre-B andmature B lymphocytes (Valentine et al. J. Biol. Chem.264(19):11282-11287 (1989); and Einfeld et al. EMBO J. 7(3):711-717(1988)). The antigen is also expressed on greater than 90% of B-cellnon-Hodgkin's lymphomas (NHL) (Anderson et al. Blood 63(6):1424-1433(1984)), but is not found on hematopoietic stem cells, pro-B cells,normal plasma cells or other normal tissues (Tedder et al. J. Immunol.135(2):973-979 (1985)). CD20 regulates an early step(s) in theactivation process for cell cycle initiation and differentiation (Tedderet al., supra) and possibly functions as a calcium ion channel (Tedderet al. J. Cell. Biochem. 14D:195 (1990)).

Given the expression of CD20 in B-cell lymphomas, this antigen can serveas a candidate for “targeting” of such lymphomas. In essence, suchtargeting can be generalized as follows: antibodies specific to the CD20surface antigen of B cells are administered to a patient. Theseanti-CD20 antibodies specifically bind to the CD20 antigen of(ostensibly) both normal and malignant B cells; the antibody bound tothe CD20 surface antigen may lead to the destruction and depletion ofneoplastic B cells. Additionally, chemical agents or radioactive labelshaving the potential to destroy the tumor can be conjugated to theanti-CD20 antibody such that the agent is specifically “delivered” tothe neoplastic B cells. Irrespective of the approach, a primary goal isto destroy the tumor; the specific approach can be determined by theparticular anti-CD20 antibody that is utilized and, thus, the availableapproaches to targeting the CD20 antigen can vary considerably.

The Rituximab (RITUXAN®) antibody is a genetically engineered chimericmurine/human monoclonal antibody directed against the CD20 antigen.Rituximab is the antibody called “C2B8” in U.S. Pat. No. 5,736,137issued Apr. 7, 1998 (Anderson et al.). RITUXAN® is indicated for thetreatment of patients with relapsed or refractory low-grade orfollicular, CD20-positive, B-cell non-Hodgkin's lymphoma. In vitromechanism of action studies have demonstrated that RITUXAN® binds humancomplement and lyses lymphoid B-cell lines through complement-dependentcytotoxicity (CDC) (Reff et al. Blood 83(2):435-445 (1994)).Additionally, it has significant activity in assays forantibody-dependent cellular cytotoxicity (ADCC). More recently, RITUXAN®has been shown to have anti-proliferative effects in tritiated thymidineincorporation assays and to induce apoptosis directly, while otheranti-CD19 and CD20 antibodies do not (Maloney et al. Blood 88(10):637a(1996)). Synergy between RITUXAN® and chemotherapies and toxins has alsobeen observed experimentally. In particular, RITUXAN® sensitizesdrug-resistant human B-cell lymphoma cell lines to the cytotoxic effectsof doxorubicin, CDDP, VP-16, diphtheria toxin and ricin (Demidem et al.Cancer Chemotherapy & Radiopharmaceuticals 12(3):177-186 (1997)). Invivo preclinical studies have shown that RITUXAN® depletes B cells fromthe peripheral blood, lymph nodes, and bone marrow of cynomolgusmonkeys, presumably through complement and cell-mediated processes (Reffet al. Blood 83(2):435-445 (1994)).

Rituximab was approved in the United States in November 1997 for thetreatment of patients with relapsed or refractory low-grade orfollicular CD20⁺ B-cell non-Hodgkin's lymphoma (NHL) at a dose of 375mg/m² weekly for four doses. In April 2001, the Food and DrugAdministration (FDA) approved additional claims for the treatment oflow-grade NHL: retreatment (weekly for four doses) and an additionaldosing regimen (weekly for eight doses). There have been more than300,000 patient exposures to Rituximab either as monotherapy or incombination with immunosuppressant or chemotherapeutic drugs. Patientshave also been treated with Rituximab as maintenance therapy for up to 2years (Hainsworth et al. J Clin Oncol 21:1746-51 (2003); Hainsworth etal. J Clin Oncol 20:4261-7 (2002)).

Rituximab has also been studied in a variety of non-malignant autoimmunedisorders, in which B cells and autoantibodies appear to play a role indisease pathophysiology (Edwards et al. Biochem Soc Trans 30:824-8(2002)). Rituximab has been reported to potentially relieve signs andsymptoms of rheumatoid arthritis (RA) (Leandro et al. Ann Rheum Dis.61:883-8 (2002); Emery et al. Arthritis Rheum 48(9):S439 (2003)), lupus(Eisenberg R. Arthritis Res Ther 5:157-9 (2003); Leandro et al.Arthritis Rheum 46:2673-7 (2002)), immune thrombocytopenia (D'Arena etal. Leuk Lymphoma 44:561-2 (2003)), autoimmune anemia (Zaja et al.Haematologica 87:189-95 (2002) (erratum appears in Haematologica 87:336(2002)), autoimmune neuropathy (Pestronk et al. J Neurol NeurosurgPsychiatry 74:485-9 (2003)), paraneoplastic opsoclonus-myoclonussyndrome (Pranzatelli et al. Neurology 60(Suppl1) P05.128:A395 (2003)),and relapsing-remitting multiple sclerosis (RRMS) (Cross et al.(abstract) Eighth Annual Meeting of the Americas Committees for Researchand Treatment in Multiple Sclerosis 20-1 (2003)).

A Phase II study (WA16291) has been conducted in patients withrheumatoid arthritis (RA), providing 48-week follow-up data on safetyand efficacy of Rituximab (Emery et al. Arthritis Rheum 48(9):5439(2003); Szczepanski et al. Arthritis Rheum 48(9):5121 (2003)). A totalof 161 patients were evenly randomized to four treatment arms:methotrexate, Rituximab alone, Rituximab plus methotrexate, Rituximabplus cyclophosphamide (CTX). The treatment regimen of Rituximab was 1 gadministered intravenously on Days 1 and 15. Infusions of Rituximab inmost patients with RA were well tolerated by most patients, with 36% ofpatients experiencing at least one adverse event during their firstinfusion (compared with 30% of patients receiving placebo). Overall, themajority of adverse events were considered to be mild to moderate inseverity and were well balanced across all treatment groups. There werea total of 19 serious adverse events across the four arms over the 48weeks, which were slightly more frequent in the Rituximab/CTX group. Theincidence of infections was well balanced across all groups. The meanrate of serious infection in this RA patient population was 4.6 6 per100 patient-years, which is lower than the rate of infections requiringhospital admission in RA patients (9.57 per 100 patient-years) reportedin a community-based epidemiologic study (Doran et al. Arthritis Rheum46:2287-93 (2002)).

The reported safety profile of Rituximab in a small number of patientswith neurologic disorders, including autoimmune neuropathy (Pestronk etal. J Neurol Neurosurg Psychiatry 74:485-9 (2003)), opsoclonus/myoclonussyndrome (Pranzatelli et al. Neurology 60(Suppl1) P05.128:A395 (2003)),and RRMS (Cross et al. Preliminary results from a phase II trial ofRituximab in MS (abstract) Eighth Annual Meeting of the AmericasCommittees for Research and Treatment in Multiple Sclerosis 20-1(2003)), was reported. In an ongoing investigator-sponsored trial (IST)of Rituximab in combination with interferon-beta (IFN-beta□) orglatiramer acetate in subjects with RRMS (Cross et al., supra), 1 of 10treated subjects was admitted to the hospital for overnight observationafter experiencing moderate fever and rigors following the firstinfusion of Rituximab, while the other 9 subjects completed thefour-infusion regimen without any reported adverse events.

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Certain of these include, inter alia, treatment of multiplesclerosis.

Publications concerning therapy with Rituximab include: Perotta andAbuel “Response of chronic relapsing ITP of 10 years duration toRituximab” Abstract #3360 Blood 10(1)(part 1-2): p. 88B (1998); Stashiet al. “Rituximab chimeric anti-CD20 monoclonal antibody treatment foradults with chronic idopathic thrombocytopenic purpura” Blood98(4):952-957 (2001); Matthews, R. “Medical Heretics” New Scientist (7April, 2001); Leandro et al. “Clinical outcome in 22 patients withrheumatoid arthritis treated with B lymphocyte depletion” Ann Rheum Dis61:833-888 (2002); Leandro et al. “Lymphocyte depletion in rheumatoidarthritis: early evidence for safety, efficacy and dose response.Arthritis and Rheumatism 44(9): 5370 (2001); Leandro et al. “An openstudy of B lymphocyte depletion in systemic lupus erythematosus”,Arthritis & Rheumatism 46(1):2673-2677 (2002); Edwards and Cambridge“Sustained improvement in rheumatoid arthritis following a protocoldesigned to deplete B lymphocytes” Rhematology 40:205-211 (2001);Edwards et al. “B-lymphocyte depletion therapy in rheumatoid arthritisand other autoimmune disorders” Biochem. Soc. Trans. 30(4):824-828(2002); Edwards et al. “Efficacy and safety of Rituximab, a B-celltargeted chimeric monoclonal antibody: A randomized, placebo controlledtrial in patients with rheumatoid arthritis. Arthritis and Rheumatism46(9): S197 (2002); Levine and Pestronk “IgM antibody-relatedpolyneuropathies: B-cell depletion chemotherapy using Rituximab”Neurology 52: 1701-1704 (1999); DeVita et al. “Efficacy of selective Bcell blockade in the treatment of rheumatoid arthritis” Arthritis &Rheum 46:2029-2033 (2002); Hidashida et al. “Treatment ofDMARD-Refractory rheumatoid arthritis with Rituximab.” Presented at theAnnual Scientific Meeting of the American College of Rheumatology;October 24-29; New Orleans, L A 2002; Tuscano, J. “Successful treatmentof Infliximab-refractory rheumatoid arthritis with Rituximab” Presentedat the Annual Scientific Meeting of the American College ofRheumatology; October 24-29; New Orleans, L A 2002; Specks et al.“Response of Wegener's granulomatosis to anti-CD20 chimeric monoclonalantibody therapy” Arthritis & Rheumatism 44(12):2836-2840 (2001); Anoliket al., “B lympocyte Depletion in the Treatment of Systemic Lupus (SLE):Phase I/II Trial of Rituximab (RITUXAN®) in SLE” Arthritis AndRheumatism, 46(9), S289-S289 Abstract 717 (October, 2002), and Albert etal., “A Phase I Trial of Rituximab (Anti-CD20) for Treatment of SystemicLupus Erythematosus” Arthritis And Rheumatism, 48(12): 3659-3659,Abstract LB9 (December, 2003); Martin and Chan “Pathogenic Roles of Bcells in Human Autoimmunity: Insights from the Clinic” Immunity20:517-527 (2004); Cree et al. “An open label study of the effects ofrituximab in neuromyelitis optica.” Neurology 64(7):1270-2 (2005); Crosset al. “Rituximab reduces B cells and T cells in cerebrospinal fluid ofmultiple sclerosis patients.” J Neuroimmunol, 180(1-2):63-70 (2006);Bar-Or A. et al., “Safety, pharmacodynamics, and activity of Rituximabin patients with relapsing-remitting multiple sclerosis: a phase I,multicentre, open-label clinical trial.” Ann Neurol 63(3):395-400(2008); Hauser S. et al., “B-cell depletion with Rituximab inrelapsing-remitting multiple sclerosis.” NEJM, 358(7):676-88, (2008);Hawker K et al., “Efficacy and Safety of rituximab in patients withprimary progressive multiple sclerosis: results of a randomized,double-blind, placebo-controlled, multicenter trial.” Multiple Sclerosis14(1):5299 (2008), Abstract; Hawker K et al., “Efficacy and Safety ofrituximab in patients with primary progressive multiple sclerosis:results of a randomized, double-blind, placebo-controlled, multicentertrial.” Neurology 72(S3):A254 (2009), Abstract.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of treating progressive multiplesclerosis in a patient comprising administering to the patient aneffective amount of an anti-CD20 antibody, wherein treatment is basedupon the patient having one or more characteristics selected from thegroup consisting of (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, (c) at least about a one point increase inExpanded Disability Status Scale (EDSS) over two years prior to startingtreatment, and (d) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points.

In some embodiments, the progressive multiple sclerosis is primaryprogressive multiple sclerosis. In some embodiments, the progressivemultiple sclerosis is secondary progressive multiple sclerosis. In someembodiments, the progressive multiple sclerosis is progressive relapsingmultiple sclerosis. In some embodiments, the patient is not diagnosedwith relapsing remitting multiple sclerosis when starting treatment.

In some embodiments, the patient further has evidence of inflammation ina sample. In some embodiments, the sample is a cerebrospinal fluidsample. In some embodiments, the evidence of inflammation is indicatedby an elevated IgG index. In some embodiments, the evidence ofinflammation is indicated by IgG oligoclonal bands detected byisoelectric focusing.

In some embodiments, the patient has had an EDSS of greater than about5.0 for less than about 15 years. In some embodiments, the patient hashad an EDSS less than or equal to about 5.0 for less than about 10years. In some embodiments, the increase in EDSS over two years prior tostarting treatment is not attributable to relapse. In some embodiments,the increase in EDSS is at least about a 1.5 point increase in EDSS overtwo years prior to starting treatment. In some embodiments, the at leastabout a 1.5 point increase in EDSS over two years prior to startingtreatment is not attributable to relapse. In some embodiments, thepatient further had two or more relapses within two years prior tostarting treatment. In some embodiments, the EDSS when startingtreatment is between about 3.0 and about 6.5.

In some embodiments, the age of the patient is less than about 51.

In some embodiments, the treatment reduces the time to confirmed diseaseprogression. In some embodiments, the confirmed disease progression isan increase in EDSS that is sustained for twelve weeks. In someembodiments, the confirmed disease progression is an increase in EDSSthat is sustained for twenty-four weeks.

In some embodiments, the effective amount of the anti-CD20 antibody isadministered to the patient to provide an initial anti-CD20 antibodyexposure of between about 0.3 to about 4.0 grams followed by a secondanti-CD20 antibody exposure of between about 0.3 to about 4.0 grams. Insome embodiments, the initial anti-CD20 antibody exposure and/or thesecond anti-CD20 antibody exposure is between about 0.3 to about 1.5grams. In some embodiments, the second exposure not being provided untilfrom about 16 to 60 weeks from the initial exposure. In someembodiments, each of the anti-CD20 antibody exposures is provided to thepatient as one or two doses of anti-CD20 antibody.

In some embodiments, the anti-CD20 antibody comprises: a) a heavy chainvariable region comprising SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12,and b) a light chain variable region comprising SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody isocrelizumab. In some embodiments, the anti-CD20 antibody is rituximab.In some embodiments, the anti-CD20 antibody is ofatumumab. In someembodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In someembodiments, the anti-CD20 antibody is GA101. In some embodiments, theanti-CD20 antibody is hA20.

The present invention provides methods of treating progressive multiplesclerosis in a patient provided that the patient has been found to haveone or more characteristics selected from the group consisting of (a) anage less than about 55 years, (b) one or more gadolinium staininglesions, (c) at least about a one point increase in Expanded DisabilityStatus Scale (EDSS) over two years prior to starting treatment, and (d)a Multiple Sclerosis Severity Score (MSSS) greater than about 5 points,the treatment comprising administering to the patient an effectiveamount of an anti-CD20 antibody.

In some embodiments, the progressive multiple sclerosis is primaryprogressive multiple sclerosis. In some embodiments, the progressivemultiple sclerosis is secondary progressive multiple sclerosis. In someembodiments, the progressive multiple sclerosis is progressive relapsingmultiple sclerosis. In some embodiments, the patient is not diagnosedwith relapsing remitting multiple sclerosis when starting treatment.

In some embodiments, the patient further has evidence of inflammation ina sample. In some embodiments, the sample is a cerebrospinal fluidsample. In some embodiments, the evidence of inflammation is indicatedby an elevated IgG index. In some embodiments, the evidence ofinflammation is indicated by IgG oligoclonal bands detected byisoelectric focusing.

In some embodiments, the patient has had an EDSS of greater than about5.0 for less than about 15 years. In some embodiments, the patient hashad an EDSS less than or equal to about 5.0 for less than about 10years. In some embodiments, the increase in EDSS over two years prior tostarting treatment is not attributable to relapse. In some embodiments,the increase in EDSS is at least about a 1.5 point increase in EDSS overtwo years prior to starting treatment. In some embodiments, the at leastabout a 1.5 point increase in EDSS over two years prior to startingtreatment is not attributable to relapse. In some embodiments, thepatient further had two or more relapses within two years prior tostarting treatment. In some embodiments, the EDSS when startingtreatment is between about 3.0 and about 6.5.

In some embodiments, the age of the patient is less than about 51.

In some embodiments, the treatment reduces the time to confirmed diseaseprogression. In some embodiments, the confirmed disease progression isan increase in EDSS that is sustained for twelve weeks. In someembodiments, the confirmed disease progression is an increase in EDSSthat is sustained for twenty-four weeks.

In some embodiments, the effective amount of the anti-CD20 antibody isadministered to the patient to provide an initial anti-CD20 antibodyexposure of between about 0.3 to about 4.0 grams followed by a secondanti-CD20 antibody exposure of between about 0.3 to about 4.0 grams. Insome embodiments, the initial anti-CD20 antibody exposure and/or thesecond anti-CD20 antibody exposure is between about 0.3 to about 1.5grams. In some embodiments, the second exposure not being provided untilfrom about 16 to 60 weeks from the initial exposure. In someembodiments, each of the anti-CD20 antibody exposures is provided to thepatient as one or two doses of anti-CD20 antibody.

In some embodiments, the anti-CD20 antibody comprises: a) a heavy chainvariable region comprising SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12,and b) a light chain variable region comprising SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody isocrelizumab. In some embodiments, the anti-CD20 antibody is rituximab.In some embodiments, the anti-CD20 antibody is ofatumumab. In someembodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In someembodiments, the anti-CD20 antibody is GA101. In some embodiments, theanti-CD20 antibody is hA20.

The present invention provides methods of treating progressive multiplesclerosis, comprising: (a) selecting a patient having progressivemultiple sclerosis, wherein said patient has one or more characteristicsselected from the group consisting of (i) an age less than about 55years, (ii) one or more gadolinium staining lesions, (iii) at leastabout a one point increase in Expanded Disability Status Scale (EDSS)over two years prior to starting treatment, and (iv) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points; and (b)administering to the patient thus selected an effective amount of ananti-CD20 antibody.

In some embodiments, the progressive multiple sclerosis is primaryprogressive multiple sclerosis. In some embodiments, the progressivemultiple sclerosis is secondary progressive multiple sclerosis. In someembodiments, the progressive multiple sclerosis is progressive relapsingmultiple sclerosis. In some embodiments, the patient is not diagnosedwith relapsing remitting multiple sclerosis when starting treatment.

In some embodiments, the patient further has evidence of inflammation ina sample. In some embodiments, the sample is a cerebrospinal fluidsample. In some embodiments, the evidence of inflammation is indicatedby an elevated IgG index. In some embodiments, the evidence ofinflammation is indicated by IgG oligoclonal bands detected byisoelectric focusing.

In some embodiments, the patient has had an EDSS of greater than about5.0 for less than about 15 years. In some embodiments, the patient hashad an EDSS less than or equal to about 5.0 for less than about 10years. In some embodiments, the increase in EDSS over two years prior tostarting treatment is not attributable to relapse. In some embodiments,the increase in EDSS is at least about a 1.5 point increase in EDSS overtwo years prior to starting treatment. In some embodiments, the at leastabout a 1.5 point increase in EDSS over two years prior to startingtreatment is not attributable to relapse. In some embodiments, thepatient further had two or more relapses within two years prior tostarting treatment. In some embodiments, the EDSS when startingtreatment is between about 3.0 and about 6.5.

In some embodiments, the age of the patient is less than about 51.

In some embodiments, the treatment reduces the time to confirmed diseaseprogression. In some embodiments, the confirmed disease progression isan increase in EDSS that is sustained for twelve weeks. In someembodiments, the confirmed disease progression is an increase in EDSSthat is sustained for twenty-four weeks.

In some embodiments, the effective amount of the anti-CD20 antibody isadministered to the patient to provide an initial anti-CD20 antibodyexposure of between about 0.3 to about 4.0 grams followed by a secondanti-CD20 antibody exposure of between about 0.3 to about 4.0 grams. Insome embodiments, the initial anti-CD20 antibody exposure and/or thesecond anti-CD20 antibody exposure is between about 0.3 to about 1.5grams. In some embodiments, the second exposure not being provided untilfrom about 16 to 60 weeks from the initial exposure. In someembodiments, each of the anti-CD20 antibody exposures is provided to thepatient as one or two doses of anti-CD20 antibody.

In some embodiments, the anti-CD20 antibody comprises: a) a heavy chainvariable region comprising SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12,and b) a light chain variable region comprising SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody isocrelizumab. In some embodiments, the anti-CD20 antibody is rituximab.In some embodiments, the anti-CD20 antibody is ofatumumab. In someembodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In someembodiments, the anti-CD20 antibody is GA101. In some embodiments, theanti-CD20 antibody is hA20.

The present invention also provides methods of assessing whether apatient with progressive multiple sclerosis will respond to treatmentwith an anti-CD20 antibody comprising assessing one or morecharacteristics selected from the group consisting of (a) an age lessthan about 55 years, (b) one or more gadolinium staining lesions, (c) atleast about a one point increase in Expanded Disability Status Scale(EDSS) over two years prior to starting treatment, and (d) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points, wherein oneor more of the characteristics in the patient indicates the patient willbe responsive to the treatment.

In some embodiments, the progressive multiple sclerosis is primaryprogressive multiple sclerosis. In some embodiments, the progressivemultiple sclerosis is secondary progressive multiple sclerosis. In someembodiments, the progressive multiple sclerosis is progressive relapsingmultiple sclerosis. In some embodiments, the patient is not diagnosedwith relapsing remitting multiple sclerosis when starting treatment.

In some embodiments, the patient further has evidence of inflammation ina sample. In some embodiments, the sample is a cerebrospinal fluidsample. In some embodiments, the evidence of inflammation is indicatedby an elevated IgG index. In some embodiments, the evidence ofinflammation is indicated by IgG oligoclonal bands detected byisoelectric focusing.

In some embodiments, the patient has had an EDSS of greater than about5.0 for less than about 15 years. In some embodiments, the patient hashad an EDSS less than or equal to about 5.0 for less than about 10years. In some embodiments, the increase in EDSS over two years prior tostarting treatment is not attributable to relapse. In some embodiments,the increase in EDSS is at least about a 1.5 point increase in EDSS overtwo years prior to starting treatment. In some embodiments, the at leastabout a 1.5 point increase in EDSS over two years prior to startingtreatment is not attributable to relapse. In some embodiments, thepatient further had two or more relapses within two years prior tostarting treatment. In some embodiments, the EDSS when startingtreatment is between about 3.0 and about 6.5.

In some embodiments, the age of the patient is less than about 51.

In some embodiments, the method further comprises advising a patient.

In some embodiments, the anti-CD20 antibody comprises: a) a heavy chainvariable region comprising SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12,and b) a light chain variable region comprising SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody isocrelizumab. In some embodiments, the anti-CD20 antibody is rituximab.In some embodiments, the anti-CD20 antibody is ofatumumab. In someembodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In someembodiments, the anti-CD20 antibody is GA101. In some embodiments, theanti-CD20 antibody is hA20.

The present invention also provides methods of identifying a patientwith progressive multiple sclerosis likely to respond to anti-CD20antibody treatment comprising: (a) assessing one or more characteristicsselected from the group consisting of (i) an age less than about 55years, (ii) one or more gadolinium staining lesions, (iii) at leastabout a one point increase in Expanded Disability Status Scale (EDSS)over two years prior to starting treatment, and (iv) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points; and (b)identifying the patient having one or more characteristics selected fromthe group consisting of (i) an age less than about 55 years, (i) one ormore gadolinium staining lesions, (iii) at least about a one pointincrease in Expanded Disability Status Scale (EDSS) over two years priorto starting the anti-CD20 treatment, and (iv) a Multiple SclerosisSeverity Score (MSSS) greater than about 5 points.

In some embodiments, the progressive multiple sclerosis is primaryprogressive multiple sclerosis. In some embodiments, the progressivemultiple sclerosis is secondary progressive multiple sclerosis. In someembodiments, the progressive multiple sclerosis is progressive relapsingmultiple sclerosis. In some embodiments, the patient is not diagnosedwith relapsing remitting multiple sclerosis when starting treatment.

In some embodiments, the patient further has evidence of inflammation ina sample. In some embodiments, the sample is a cerebrospinal fluidsample. In some embodiments, the evidence of inflammation is indicatedby an elevated IgG index. In some embodiments, the evidence ofinflammation is indicated by IgG oligoclonal bands detected byisoelectric focusing.

In some embodiments, the patient has had an EDSS of greater than about5.0 for less than about 15 years. In some embodiments, the patient hashad an EDSS less than or equal to about 5.0 for less than about 10years. In some embodiments, the increase in EDSS over two years prior tostarting treatment is not attributable to relapse. In some embodiments,the increase in EDSS is at least about a 1.5 point increase in EDSS overtwo years prior to starting treatment. In some embodiments, the at leastabout a 1.5 point increase in EDSS over two years prior to startingtreatment is not attributable to relapse. In some embodiments, thepatient further had two or more relapses within two years prior tostarting treatment. In some embodiments, the EDSS when startingtreatment is between about 3.0 and about 6.5.

In some embodiments, the age of the patient is less than about 51.

In some embodiments, the method further comprises advising a patient.

In some embodiments, the anti-CD20 antibody comprises: a) a heavy chainvariable region comprising SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12,and b) a light chain variable region comprising SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody isocrelizumab. In some embodiments, the anti-CD20 antibody is rituximab.In some embodiments, the anti-CD20 antibody is ofatumumab. In someembodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In someembodiments, the anti-CD20 antibody is GA101. In some embodiments, theanti-CD20 antibody is hA20.

The present invention further provides methods for marketing ananti-CD20 antibody or a pharmaceutically acceptable composition thereoffor use in a progressive multiple sclerosis patient subpopulation, themethods comprising informing a target audience about the use of theanti-CD20 antibody for treating the patient subpopulation characterizedby the patients of such subpopulation having one or more characteristicsselected from the group consisting of (a) an age less than about 55years, (b) one or more gadolinium staining lesions, (c) at least about aone point increase in Expanded Disability Status Scale (EDSS) over twoyears prior to starting treatment, and (d) a Multiple Sclerosis SeverityScore (MSSS) greater than about 5 points.

In some embodiments, the progressive multiple sclerosis is primaryprogressive multiple sclerosis. In some embodiments, the progressivemultiple sclerosis is secondary progressive multiple sclerosis. In someembodiments, the progressive multiple sclerosis is progressive relapsingmultiple sclerosis. In some embodiments, the patient subpopulation isnot diagnosed with relapsing remitting multiple sclerosis when startingtreatment.

In some embodiments, the patient subpopulation further has evidence ofinflammation in a sample. In some embodiments, the sample is acerebrospinal fluid sample. In some embodiments, the evidence ofinflammation is indicated by an elevated IgG index. In some embodiments,the evidence of inflammation is indicated by IgG oligoclonal bandsdetected by isoelectric focusing.

In some embodiments, the patient subpopulation had an EDSS of greaterthan about 5.0 for less than about 15 years. In some embodiments, thepatient subpopulation had an EDSS less than or equal to about 5.0 forless than about 10 years. In some embodiments, the increase in EDSS overtwo years prior to starting treatment is not attributable to relapse. Insome embodiments, the increase in EDSS is at least about a 1.5 pointincrease in EDSS over two years prior to starting treatment. In someembodiments, the at least about a 1.5 point increase in EDSS over twoyears prior to starting treatment is not attributable to relapse. Insome embodiments, the patient subpopulation further had two or morerelapses within two years prior to starting treatment. In someembodiments, the EDSS when starting treatment is between about 3.0 andabout 6.5.

In some embodiments, the age of the patient subpopulation is less thanabout 51.

In some embodiments, the anti-CD20 antibody comprises: a) a heavy chainvariable region comprising SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12,and b) a light chain variable region comprising SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody isocrelizumab. In some embodiments, the anti-CD20 antibody is rituximab.In some embodiments, the anti-CD20 antibody is ofatumumab. In someembodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In someembodiments, the anti-CD20 antibody is GA101. In some embodiments, theanti-CD20 antibody is hA20.

The present invention provides articles of manufacture comprising,packaged together, a pharmaceutical composition comprising an anti-CD20antibody and a pharmaceutically acceptable carrier and a label denoting(i.e., indicating) that the anti-CD20 antibody or pharmaceuticalcomposition is indicated for treating patients with multiple sclerosishaving one or more characteristics selected from the group consisting of(a) an age less than about 55 years, (b) one or more gadolinium staininglesions, (c) at least about a one point increase in Expanded DisabilityStatus Scale (EDSS) over two years prior to starting the treatment, and(d) a Multiple Sclerosis Severity Score (MSSS) greater than about 5points.

In some embodiments, the progressive multiple sclerosis is primaryprogressive multiple sclerosis. In some embodiments, the progressivemultiple sclerosis is secondary progressive multiple sclerosis. In someembodiments, the progressive multiple sclerosis is progressive relapsingmultiple sclerosis. In some embodiments, the patient is not diagnosedwith relapsing remitting multiple sclerosis when starting treatment.

In some embodiments, the patient further has evidence of inflammation ina sample. In some embodiments, the sample is a cerebrospinal fluidsample. In some embodiments, the evidence of inflammation is indicatedby an elevated IgG index. In some embodiments, the evidence ofinflammation is indicated by IgG oligoclonal bands detected byisoelectric focusing.

In some embodiments, the patient has had an EDSS of greater than about5.0 for less than about 15 years. In some embodiments, the patient hashad an EDSS less than or equal to about 5.0 for less than about 10years. In some embodiments, the increase in EDSS over two years prior tostarting treatment is not attributable to relapse. In some embodiments,the increase in EDSS is at least about a 1.5 point increase in EDSS overtwo years prior to starting treatment. In some embodiments, the at leastabout a 1.5 point increase in EDSS over two years prior to startingtreatment is not attributable to relapse. In some embodiments, thepatient further had two or more relapses within two years prior tostarting treatment. In some embodiments, the EDSS when startingtreatment is between about 3.0 and about 6.5.

In some embodiments, the age of the patient is less than about 51.

In some embodiments, the pharmaceutical composition comprising theanti-CD20 antibody and the pharmaceutically acceptable carrier is in acontainer. In some embodiments, the container comprises between about0.3 to about 4.0 grams of the anti-CD20 antibody. In some embodiments,the container comprises between about 0.3 to about 1.5 grams of theanti-CD20 antibody.

In some embodiments, the label provides instructions, wherein theinstructions indicate that an effective amount of the anti-CD20 antibodyis administered to the patient to provide an initial anti-CD20 antibodyexposure of between about 0.3 to about 4.0 grams followed by a secondanti-CD20 antibody exposure of between about 0.3 to about 4.0 grams. Insome embodiments, the initial anti-CD20 antibody exposure and/or thesecond anti-CD20 antibody exposure is between about 0.3 to about 1.5grams. In some embodiments, the second exposure not being provided untilfrom about 16 to 60 weeks from the initial exposure. In someembodiments, each of the anti-CD20 antibody exposures is provided to thepatient as one or two doses of anti-CD20 antibody.

In some embodiments, the anti-CD20 antibody comprises: a) a heavy chainvariable region comprising SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12,and b) a light chain variable region comprising SEQ ID NO:4, SEQ IDNO:5, and SEQ ID NO:6. In some embodiments, the anti-CD20 antibody isocrelizumab. In some embodiments, the anti-CD20 antibody is rituximab.In some embodiments, the anti-CD20 antibody is ofatumumab. In someembodiments, the anti-CD20 antibody is TRU-015 or SBI-087. In someembodiments, the anti-CD20 antibody is GA101. In some embodiments, theanti-CD20 antibody is hA20.

The present invention also provides methods for predicting whether asubject with progressive multiple sclerosis will respond to a treatmentwith a drug used to treat multiple sclerosis, the methods comprisingassessing one or more characteristics selected from the group consistingof (a) an age less than about 55 years, (b) one or more gadoliniumstaining lesions, (c) at least about a one point increase in ExpandedDisability Status Scale (EDSS) over two years prior to startingtreatment, and (d) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points, whereby the age, the gadolinium staining lesions,the increase in EDDS over two years prior to starting the treatment, theMSSS, or a combination thereof indicates that the subject will respondto the treatment.

In some embodiments, the progressive multiple sclerosis is primaryprogressive multiple sclerosis. In some embodiments, the progressivemultiple sclerosis is secondary progressive multiple sclerosis. In someembodiments, the progressive multiple sclerosis is progressive relapsingmultiple sclerosis. In some embodiments, the subject is not diagnosedwith relapsing remitting multiple sclerosis when starting treatment.

In some embodiments, the subject further has had evidence ofinflammation in a sample. In some embodiments, the sample is acerebrospinal fluid sample. In some embodiments, the evidence ofinflammation is indicated by an elevated IgG index. In some embodiments,the evidence of inflammation is indicated by IgG oligoclonal bandsdetected by isoelectric focusing.

In some embodiments, the subject has had an EDSS of greater than about5.0 for less than about 15 years. In some embodiments, the subject hadan EDSS less than or equal to about 5.0 for less than about 10 years. Insome embodiments, the increase in EDSS over two years prior to startingtreatment is not attributable to relapse. In some embodiments, theincrease in EDSS is at least about a 1.5 point increase in EDSS over twoyears prior to starting treatment. In some embodiments, the at leastabout a 1.5 point increase in EDSS over two years prior to startingtreatment is not attributable to relapse. In some embodiments, thesubject further had two or more relapses within two years prior tostarting treatment. In some embodiments, the EDSS when startingtreatment is between about 3.0 and about 6.5.

In some embodiments, the age of the subject is less than about 51.

In some embodiments of any of the methods or articles of manufacturedescribed herein, the patients have one or more characteristics selectedfrom the group consisting of (a) an age less than about 55 years, (b)one or more gadolinium staining lesions, and (c) at least about a onepoint increase in Expanded Disability Status Scale (EDSS) over two yearsprior to starting treatment.

The invention further provides methods of treating multiple sclerosis ina patient comprising administering an effective amount of ocrelizumab tothe patient to provide an initial ocrelizumab exposure of between about0.3 to about 0.6 grams followed by a second ocrelizumab exposure ofbetween about 0.3 to about 0.6 grams, the second exposure not beingprovided until from about 16 to 60 weeks from the initial exposure, andeach of the ocrelizumab exposures is provided to the patient as one ortwo doses of ocrelizumab.

In some embodiments, the initial ocrelizumab exposure is about 0.6grams. In some embodiments, the second ocrelizumab exposure is about 0.6grams. In some embodiments, the second exposure is administered fromabout 24 weeks from the initial exposure. In some embodiments, one ormore of the ocrelizumab exposures are provided to the patient as onedose of ocrelizumab. In some embodiments, one or more of the ocrelizumabexposures are provided to the patient as two doses of ocrelizumab. Insome embodiments, the initial ocrelizumab exposure comprises a firstdose and a second dose of ocrelizumab, wherein the first dose and seconddose of ocrelizumab is about 0.3 grams. In some embodiments, the secondocrelizumab exposure comprises a single dose of ocrelizumab, wherein thesingle dose of ocrelizumab is 0.6 grams. In some embodiments, themethods further comprising providing a third ocrelizumab exposure. Insome embodiments, the methods further comprising providing a fourthocrelizumab exposure. In some embodiments, the methods furthercomprising providing a fifth ocrelizumab exposure. In some embodimentsof any of the methods, the methods further comprising providing betweenabout one to about three subsequent ocrelizumab exposures.

The invention also provides articles of manufacture comprising: (a) acontainer comprising ocrelizumab; and (b) a package insert withinstructions for treating multiple sclerosis in a patient, wherein theinstructions denote that an amount of ocrelizumab is administered to thepatient that is effective to provide an initial ocrelizumab exposure ofbetween about 0.3 to about 0.6 grams followed by a second ocrelizumabexposure of between about 0.3 to about 0.6 grams, the second exposurenot being administered until from about 16 to 60 weeks from the initialexposure, and each of the ocrelizumab exposures is provided to thepatient as one or two doses of ocrelizumab.

In some embodiments, the initial ocrelizumab exposure is about 0.6grams. In some embodiments, the second ocrelizumab exposure is about 0.6grams. In some embodiments, the second exposure is administered fromabout 24 weeks from the initial exposure. In some embodiments, one ormore of the ocrelizumab exposures are provided to the patient as onedose of ocrelizumab. In some embodiments, one or more of the ocrelizumabexposures are provided to the patient as two doses of ocrelizumab. Insome embodiments, the initial ocrelizumab exposure comprises a firstdose and a second dose of ocrelizumab, wherein the first dose and seconddose of ocrelizumab is about 0.3 grams. In some embodiments, theinstructions further comprising providing a third ocrelizumab exposure.In some embodiments, the instructions further comprising providing afourth ocrelizumab exposure. In some embodiments, the instructionsfurther comprising providing a fifth ocrelizumab exposure. In someembodiments of any of the methods, the instructions further comprisingproviding between about one to about three subsequent ocrelizumabexposures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a sequence alignment comparing the amino acid sequences ofthe light chain variable domain (V_(L)) of each of murine 2H7 (SEQ IDNO:1), humanized 2H7.v16 variant (SEQ ID NO:2), and the human kappalight chain subgroup I (SEQ ID NO:3). The CDRs of V_(L) of 2H7 andhu2H7.v16 are as follows: CDR1 (SEQ ID NO:4), CDR2 (SEQ ID NO:5), andCDR3 (SEQ ID NO:6).

FIG. 1B is a sequence alignment comparing the amino acid sequences ofthe heavy chain variable domain (V_(H)) of each of murine 2H7 (SEQ IDNO:7), humanized 2H7.v16 variant (SEQ ID NO:8), and the human consensussequence of the heavy chain subgroup III (SEQ ID NO:9). The CDRs ofV_(H) of 2H7 and hu2H7.v16 are as follows: CDR1 (SEQ ID NO:10), CDR2(SEQ ID NO:11), and CDR3 (SEQ ID NO:12).

In FIG. 1A and FIG. 1B, the CDR1, CDR2 and CDR3 in each chain areenclosed within brackets, flanked by the framework regions, FR1-FR4, asindicated. 2H7 refers to the murine 2H7 antibody. The asterisks inbetween two rows of sequences indicate the positions that are differentbetween the two sequences. Residue numbering is according to Kabat etal. Sequences of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), with insertionsshown as a, b, c, d, and e.

FIG. 2 shows the amino acid sequence of the mature 2H7.v16 light chain(SEQ ID NO:13)

FIG. 3 shows the amino acid sequence of the mature 2H7.v16 heavy chain(SEQ ID NO:14).

FIG. 4 shows the amino acid sequence of the mature 2H7.v31 heavy chain(SEQ ID NO:15). The L chain of 2H7.v31 is the same as for 2H7.v16.

FIG. 5 shows an alignment of the mature 2H7.v16 and 2H7.v511 lightchains (SEQ ID NOS. 13 and 16, respectively), with Kabat variable domainresidue numbering and Eu constant domain residue numbering.

FIG. 6 shows an alignment of the mature 2H7.v16 and 2H7.v511 heavychains (SEQ ID NOS. 14 and 17, respectively), with Kabat variable domainresidue numbering and Eu constant domain residue numbering.

FIG. 7 shows an overview of the study design for treatingrelapsing-remitting multiple sclerosis using ocrelizumab.

FIG. 8 shows Kaplan Meier plots of the time to confirmed diseaseprogression for subjects in the placebo and rituximab groups.

FIG. 9 shows the median change in T2 lesion volume from baseline to week96. The Y-axis shows the T2 Lesion Volume mm³.

FIG. 10 shows a summary of baseline characteristics and hazard ratio ofsubjects in the placebo and rituximab groups.

FIGS. 11A-11D show multivariate analysis of additive predictive effectsof age and gadolinium (Gd) lesion at baseline for the treatment effectin the placebo and rituximab groups. FIG. 11A shows multivariateanalysis of age <51 and Gd lesions at baseline=0. FIG. 11B showsmultivariate analysis of age ≥51 and Gd lesions at baseline=0. FIG. 11Cshows multivariate analysis of age <51 and Gd lesions at baseline ≥1.FIG. 11D shows multivariate analysis of age ≥51 and Gd lesions atbaseline ≥1.

FIGS. 12A-12D show multivariate analysis of additive predictive effectsof age and Multiple Sclerosis Severity Score (MSSS) for the treatmenteffect in the placebo and rituximab groups. FIG. 12A shows multivariateanalysis of age ≤55 and MSSS<5. FIG. 12B shows multivariate analysis ofage >55 and MSSS<5. FIG. 12C shows multivariate analysis of age <55 andMSSS≥5. FIG. 12D shows multivariate analysis of age >55 and MSSS≥5.

FIG. 13 shows Kaplan Meier plots for the time to confirmed diseaseprogression of subjects in the placebo and rituximab groups with thefollowing characteristics: age ≤55; 3≤baseline EDSS≤6.5; excludingpatients with disease duration >10 years if their baseline EDSS<5 ordisease duration >15 if their baseline EDSS≥5.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

A “B-cell” is a lymphocyte that matures within the bone marrow, andincludes a naive B cell, memory B cell, or effector B cell (plasmacells). The B-cell herein may be a normal or non-malignant B cell.

A “B-cell surface marker” or “B-cell surface antigen” herein is anantigen expressed on the surface of a B cell that can be targeted withan antibody that binds thereto. Exemplary B-cell surface markers includethe CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72,CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82,CD83, CDw84, CD85 and CD86 leukocyte surface markers (for descriptions,see The Leukocyte Antigen Facts Book, 2^(nd) Edition. 1997, ed. Barclayet al. Academic Press, Harcourt Brace & Co., New York). Other B-cellsurface markers include RP105, FcRH2, B-cell CR2, CCR6, P2X5, HLA-DOB,CXCRS, FCER2, BR3, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3,IRTA1, FcRH6, BCMA, and 239287. The B-cell surface marker of particularinterest herein is preferentially expressed on B cells compared to othernon-B-cell tissues of a mammal and may be expressed on both precursor Bcells and mature B cells. The preferred B-cell surface marker herein isCD20.

The “CD20” antigen, or “CD20,” is an about 35-kDa, non-glycosylatedphosphoprotein found on the surface of greater than 90% of B cells fromperipheral blood or lymphoid organs. CD20 is present on both normal Bcells as well as malignant B cells, but is not expressed on stem cells.Other names for CD20 in the literature include “B-lymphocyte-restrictedantigen” and “Bp35”. The CD20 antigen is described in Clark et al. Proc.Natl. Acad. Sci. (USA) 82:1766 (1985), for example.

An “antibody antagonist” herein is a antibody that, upon binding to a Bcell surface marker on B cells, destroys or depletes B cells in a mammaland/or interferes with one or more B-cell functions, e.g. by reducing orpreventing a humoral response elicited by the B cell. The antibodyantagonist preferably is able to deplete B cells (i.e. reducecirculating B-cell levels) in a mammal treated therewith. Such depletionmay be achieved via various mechanisms such antibody-dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC), inhibition of B-cell proliferation and/or inductionof B-cell death (e.g. via apoptosis).

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes that express one or more FcRs andperform effector functions. In some embodiments, the cells express atleast FcγRIII and carry out ADCC effector function. Examples of humanleukocytes that mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils; with PBMCs and NK cells being preferred.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. In some embodiments, the FcR is anative sequence human FcR. Moreover, a preferred FcR is one that bindsan IgG antibody (a gamma receptor) and includes receptors of the FcγRI,FcγRII, and FcγRIII subclasses, including allelic variants andalternatively spliced forms of these receptors. FcγRII receptors includeFcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitingreceptor”), which have similar amino acid sequences that differprimarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (ITIM) in itscytoplasmic domain. (see Daëron, Annu. Rev. Immunol. 15:203-234 (1997)).FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al.,J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to beidentified in the future, are encompassed by the term “FcR” herein. Theterm also includes the neonatal receptor, FcRn, which is responsible forthe transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)).

“Complement dependent cytotoxicity” or “CDC” refer to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (Clq) to a molecule (e.g. an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996), may be performed.

“Growth inhibitory” antibodies are those that prevent or reduceproliferation of a cell expressing an antigen to which the antibodybinds. For example, the antibody may prevent or reduce proliferation ofB cells in vitro and/or in vivo.

Antibodies that “induce apoptosis” are those that induce programmed celldeath, e.g. of a B cell, as determined by standard apoptosis assays,such as binding of annexin V, fragmentation of DNA, cell shrinkage,dilation of endoplasmic reticulum, cell fragmentation, and/or formationof membrane vesicles (called apoptotic bodies).

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

For the purposes herein, an “intact antibody” is one comprising heavyand light variable domains as well as an Fc region.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments that have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, antibodies can be assigned to different classes. There arefive major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM,and several of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constantdomains that correspond to the different classes of antibodies arecalled α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. In some embodiments, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the scFv to form the desired structure for antigen binding. Fora review of scFv see Plückthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they are uncontaminated by other immunoglobulins.The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature,256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).Chimeric antibodies of interest herein include “primatized” antibodiescomprising variable domain antigen-binding sequences derived from anon-human primate (e.g. Old World Monkey, such as baboon, rhesus orcynomolgus monkey) and human constant region sequences (U.S. Pat. No.5,693,780). “Humanized” forms of non-human (e.g., murine) antibodies arechimeric antibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence, except for FRsubstitution(s) as noted above. The humanized antibody optionally alsowill comprise at least a portion of an immunoglobulin constant region,typically that of a human immunoglobulin. For further details, see Joneset at, Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

A “naked antibody” is an antibody (as herein defined) that is notconjugated to a heterologous molecule, such as a cytotoxic moiety orradiolabel.

Examples of Examples of anti-CD20 antibodies include: “C2B8,” which isnow called “rituximab” (“RITUXAN®/MABTHERA®”) (U.S. Pat. No. 5,736,137);the yttrium-[90]-labelled 2B8 murine antibody designated “Y2B8” or“Ibritumomab Tiuxetan” (ZEVALIN®) commercially available from BiogenIdec, Inc. (e.g., U.S. Pat. No. 5,736,137; 2B8 deposited with ATCC underaccession no. HB11388 on Jun. 22, 1993); murine IgG2a “B1,” also called“Tositumomab,” optionally labelled with ¹³¹I to generate the “131I-B1”or “iodine I131 tositumomab” antibody (BEXXAR™) commercially availablefrom Corixa (see, also, e.g., U.S. Pat. No. 5,595,721); murinemonoclonal antibody “1F5” (e.g., Press et al. Blood 69(2):584-591 (1987)and variants thereof including “framework patched” or humanized 1F5(e.g., WO 2003/002607, Leung, S.; ATCC deposit HB-96450); murine 2H7 andchimeric 2H7 antibody (e.g., U.S. Pat. No. 5,677,180); a 2H7 antibody(e.g., WO 2004/056312 (Lowman et al.) and as set forth below);HUMAX-CD20^(Im) (ofatumumab) fully human, high-affinity antibodytargeted at the CD20 molecule in the cell membrane of B-cells (Genmab,Denmark; see, for example, Glennie and van de Winkel, Drug DiscoveryToday 8: 503-510 (2003) and Cragg et al., Blood 101: 1045-1052 (2003));the human monoclonal antibodies set forth in WO 2004/035607 and WO2005/103081 (Teeling et al., GenMab/Medarex); the antibodies havingcomplex N-glycoside-linked sugar chains bound to the Fc region describedin US 2004/0093621 (Shitara et al.); a chimerized or humanizedmonoclonal antibody having a high binding affinity to an extracellularepitope of a CD20 antigen described in WO 2006/106959 (Numazaki et al.,Biomedics Inc.); monoclonal antibodies and antigen-binding fragmentsbinding to CD20 (e.g., WO 2005/000901, Tedder et al.) such as HB20-3,HB20-4, HB20-25, and MB20-11; single-chain proteins binding to CD20including, but not limited to, TRU-015 (e.g., US 2005/0186216 (Ledbetterand Hayden-Ledbetter); US 2005/0202534 (Hayden-Ledbetter and Ledbetter);US 2005/0202028 (Hayden-Ledbetter and Ledbetter); US 2005/136049(Ledbetter et al.); US 2005/0202023 (Hayden-Ledbetter andLedbetter)—Trubion Pharm Inc.); CD20-binding molecules such as the AMEseries of antibodies, e.g., AME-133 antibodies as set forth, forexample, in WO 2004/103404; US 2005/0025764; and US 2006/0251652(Watkins et al., Applied Molecular Evolution, Inc.) and the anti-CD20antibodies with Fc mutations as set forth, for example, in WO2005/070963 (Allan et al., Applied Molecular Evolution, Inc.);CD20-binding molecules such as those described in WO 2005/016969 and US2005/0069545 (Carr et al.); bispecific antibodies as set forth, forexample, in WO 2005/014618 (Chang et al.); humanized LL2 monoclonalantibodies and other anti-CD20 antibodies as described, for example, inU.S. Pat. No. 7,151,164 (Hansen et al., Immunomedics; US 2005/0106108(Leung and Hansen; Immunomedics); fully human antibodies against CD20 asdescribed, e.g., in WO 2006/130458; Gazit et al., Amgen/AstraZeneca);antibodies against CD20 as described, for example, in WO 2006/126069(Morawala, Avestha Gengraine Technologies Pvt Ltd.); chimeric orhumanized B-Ly1 antibodies to CD20 (e.g., GA-101) as described, forexample, in WO 2005/044859; US 2005/0123546; US 2004/0072290; and US2003/0175884 (Umana et al.; GlycArt Biotechnology AG); A20 antibody orvariants thereof such as chimeric or humanized A20 antibody (cA20, hA20,respectively) and IMMUN-106 (e.g., US 2003/0219433, Immunomedics); andmonoclonal antibodies L27, G28-2, 93-1B3, B-C1 or NU-B2 available fromthe International Leukocyte Typing Workshop (e.g., Valentine et al., In:Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press(1987)). In some embodiments,the anti-CD20 antibodies herein arechimeric, humanized, or human anti-CD20 antibodies, more preferablyrituximab, a 2H7 antibody, chimeric or humanized A20 antibody(Immunomedics), and HUMAX-CD20™ human anti-CD20 antibody (Genmab).

The terms “Rituximab” or “RITUXAN®” herein refer to the geneticallyengineered chimeric murine/human monoclonal antibody directed againstthe CD20 antigen and designated “C2B8” in U.S. Pat. No. 5,736,137,including fragments thereof that retain the ability to bind CD20.Rituximab is commercially available from Genentech.

Purely for the purposes herein and unless indicated otherwise,“humanized 2H7” refers to a humanized antibody that binds human CD20, oran antigen-binding fragment thereof, wherein the antibody is effectiveto deplete primate B cells in vivo, the antibody comprising in the Hchain variable region (V_(H)) thereof at least a CDR H3 sequence of SEQID NO:12 (FIG. 1B) from an anti-human CD20 antibody and substantiallythe human consensus framework (FR) residues of the human heavy-chainsubgroup III (V_(H)III). In some embodiments, this antibody furthercomprises the H chain CDR H1 sequence of SEQ ID NO:10 and CDR H2sequence of SEQ ID NO:11, and, in some embodiments, further comprisesthe L chain CDR L1 sequence of SEQ ID NO:4, CDR L2 sequence of SEQ IDNO:5, CDR L3 sequence of SEQ ID NO:6 and substantially the humanconsensus framework (FR) residues of the human light chain □ subgroup I(V□I), wherein the V_(H) region may be joined to a human IgG chainconstant region, wherein the region may be, for example, IgG1 or IgG3.In some embodiments, such antibody comprises the V_(H) sequence of SEQID NO:8 (v16, as shown in FIG. 1B), optionally also comprising the V_(L)sequence of SEQ ID NO:2 (v16, as shown in FIG. 1A), which may have theamino acid substitutions of D56A and N100A in the H chain and S92A inthe L chain (v96). In some embodiments, the antibody is an intactantibody comprising the light and heavy chain amino acid sequences ofSEQ ID NOS:13 and 14, respectively, as shown in FIGS. 2 and 3. In someembodiments, the antibody is 2H7.v31 comprising the light and heavychain amino acid sequences of SEQ ID NOS:13 and 15, respectively, asshown in FIGS. 2 and 4. The antibody herein may further comprise atleast one amino acid substitution in the Fc region that improves ADCCand/or CDC activity, such as one wherein the amino acid substitutionsare S298A/E333A/K334A, and in some embodiments, the 2H7.v31 having theheavy chain amino acid sequence of SEQ ID NO:15 (as shown in FIG. 4).Any of these antibodies may further comprise at least one amino acidsubstitution in the Fc region that decreases CDC activity, for example,comprising at least the substitution K322A. See U.S. Pat. No.6,528,624B1 (Idusogie et al.).

The term “Ocrelizumab” herein refers to the genetically engineeredhumanized monoclonal antibody directed against the CD20 antigen andcomprising (a) a light chain comprising the amino acid sequence of SEQID NO: 13 and (b) a heavy chain comprising the amino acid sequence ofSEQ ID NO:14, including fragments thereof that retain the ability tobind CD20. Ocrelizumab is available from Genentech.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In some embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and in some embodiments, more than 99% by weight, (2)to a degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, in some embodiments, silver stain. Isolatedantibody includes the antibody in situ within recombinant cells since atleast one component of the antibody's natural environment will not bepresent. Ordinarily, however, isolated antibody will be prepared by atleast one purification step.

A “subject” or “patient” herein is a human subject or patient.Generally, the subject or patient is eligible for treatment for multiplesclerosis. For the purposes herein, such eligible subject or patient isone who is experiencing, has experienced, or is likely to experience,one or more signs, symptoms or other indicators of multiple sclerosis;has been diagnosed with multiple sclerosis, whether, for example, newlydiagnosed (with “new onset” MS), previously diagnosed with a new relapseor exacerbation, previously diagnosed and in remission, etc; and/or isat risk for developing multiple sclerosis. One suffering from or at riskfor suffering from multiple sclerosis may optionally be identified asone who has been screened for elevated levels of CD20-positive B cellsin serum, cerebrospinal fluid (CSF) and/or MS lesion(s) and/or isscreened for using an assay to detect autoantibodies, assessedqualitatively, and preferably quantitatively. Exemplary suchautoantibodies associated with multiple sclerosis include anti-myelinbasic protein (MBP), anti-myelin oligodendrocytic glycoprotein (MOG),anti-ganglioside and/or anti-neurofilament antibodies. Suchautoantibodies may be detected in the subject's serum, cerebrospinalfluid (CSF) and/or MS lesion. By “elevated” autoantibody or B celllevel(s) herein is meant level(s) of such autoantibodies or B cellswhich significantly exceed the level(s) in an individual without MS.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: decreasing one or moresymptoms resulting from the disease, diminishing the extent of thedisease, stabilizing the disease (e.g., preventing or delaying theworsening of the disease), delay or slowing the progression of thedisease, ameliorating the disease state, decreasing the dose of one ormore other medications required to treat the disease, and/or increasingthe quality of life.

As used herein, “delaying” the progression of multiple sclerosis meansto defer, hinder, slow, retard, stabilize, and/or postpone developmentof the disease. This delay can be of varying lengths of time, dependingon the history of the disease and/or individual being treated.

As used herein, “at the time of starting treatment” refers to the timeperiod at or prior to the first exposure to a multiple sclerosis drug,such as an anti-CD20 antibody. In some embodiments, “at the time ofstarting treatment” is about any of one year, nine months, six months,three months, second months, or one month prior to a multiple sclerosisdrug, such as an anti-CD20 antibody. In some embodiments, “at the timeof starting treatment” is immediately prior to coincidental with thefirst exposure to a multiple sclerosis drug, such as an anti-CD20antibody.

As used herein, “based upon” includes (1) assessing, determining, ormeasuring the patient characteristics as described herein (andpreferably selecting a patient suitable for receiving treatment; and (2)administering the treatment(s) as described herein.

A “symptom” of MS is any morbid phenomenon or departure from the normalin structure, function, or sensation, experienced by the subject andindicative of MS.

“Multiple sclerosis” refers to the chronic and often disabling diseaseof the central nervous system characterized by the progressivedestruction of the myelin. There are four internationally recognizedforms of MS, namely, primary progressive multiple sclerosis (PPMS),relapsing-remitting multiple sclerosis (RRMS), secondary progressivemultiple sclerosis (SPMS), and progressive relapsing multiple sclerosis(PRMS).

“Progressive multiple sclerosis” as used herein refers to primaryprogressive multiple sclerosis (PPMS), secondary progressive multiplesclerosis (SPMS), and progressive relapsing multiple sclerosis (PRMS).In some embodiments, progressive multiple sclerosis is characterized bydocumented, irreversible loss of neurological function persisting for ≥6months that cannot be attributed to clinical relapse.

“Primary progressive multiple sclerosis” or “PPMS” is characterized by agradual progression of the disease from its onset with no superimposedrelapses and remissions at all. There may be periods of a leveling offof disease activity and there may be good and bad days or weeks. PPMSdiffers from RRMS and SPMS in that onset is typically in the latethirties or early forties, men are as likely women to develop it, andinitial disease activity is often in the spinal cord and not in thebrain. PPMS often migrates into the brain, but is less likely to damagebrain areas than RRMS or SPMS. For example, people with PPMS are lesslikely to develop cognitive problems than those with RRMS or SPMS. PPMSis the sub-type of MS that is least likely to show inflammatory(gadolinium enhancing) lesions on MRI scans. The Primary Progressiveform of the disease affects between 10 and 15% of all people withmultiple sclerosis. PPMS may be defined according to the criteria inMcDonald et al. Ann Neurol 50:121-7 (2001). The subject with PPMStreated herein is usually one with probable or definitive diagnosis ofPPMS.

“Relapsing-remitting multiple sclerosis” or “RRMS” is characterized byrelapses (also known as exacerbations) during which time new symptomscan appear and old ones resurface or worsen. The relapses are followedby periods of remission, during which time the person fully or partiallyrecovers from the deficits acquired during the relapse. Relapses canlast for days, weeks or months and recovery can be slow and gradual oralmost instantaneous. The vast majority of people presenting with MS arefirst diagnosed with RRMS. This is typically when they are in theirtwenties or thirties, though diagnoses much earlier or later are known.Twice as many women as men present with this sub-type of MS. Duringrelapses, myelin, a protective insulating sheath around the nerve fibers(neurons) in the white matter regions of the central nervous system(CNS), may be damaged in an inflammatory response by the body's ownimmune system. This causes a wide variety of neurological symptoms thatvary considerably depending on which areas of the CNS are damaged.Immediately after a relapse, the inflammatory response dies down and aspecial type of glial cell in the CNS (called an oligodendrocyte)sponsors remyelination—a process whereby the myelin sheath around theaxon may be repaired. It is this remyelination that may be responsiblefor the remission. Approximately 50% of patients with RRMS convert toSPMS within 10 years of disease onset. After 30 years, this figure risesto 90%. At any one time, the relapsing-remitting form of the diseaseaccounts around 55% of all people with MS.

“Secondary progressive multiple sclerosis” or “SPMS” is characterized bya steady progression of clinical neurological damage with or withoutsuperimposed relapses and minor remissions and plateaux. People whodevelop SPMS will have previously experienced a period of RRMS which mayhave lasted anything from two to forty years or more. Any superimposedrelapses and remissions there are, tend to tail off over time. From theonset of the secondary progressive phase of the disease, disabilitystarts advancing much quicker than it did during RRMS though theprogress can still be quite slow in some individuals. After 10 years,50% of people with RRMS will have developed SPMS. By 25 to 30 years,that figure will have risen to 90%. SPMS tends to be associated withlower levels of inflammatory lesion formation than in RRMS but the totalburden of disease continues to progress. At any one time, SPMS accountsaround 30% of all people with multiple sclerosis.

“Progressive relapsing multiple sclerosis” refers to “PRMS” ischaracterized by a steady progression of clinical neurological damagewith superimposed relapses and remissions. There is significant recoveryimmediately following a relapse but between relapses there is a gradualworsening of symptoms. PRMS affects around 5% of all people withmultiple sclerosis. Some neurologists believe PRMS is a variant of PPMS.

The expression “effective amount” refers to an amount of the antibody(or other drug) that is effective for ameliorating or treating themultiple sclerosis. Such an effective amount will generally result in animprovement in the signs, symptoms or other indicators of MS, such asreducing relapse rate, preventing disability, reducing number and/orvolume of brain MRI lesions, improving timed 25-foot walk, slow or delaythe progression of the disease such as extending the time to diseaseprogression (e.g. using Expanded Disability Status Scale, EDSS), etc.

“Antibody exposure” refers to contact with or exposure to the antibodyherein in one or more doses administered over a period of time of about1-20 days. The doses may be given at one time or at fixed or irregulartime intervals over this period of exposure. Initial and later (e.g.second or third) antibody exposures are separated in time from eachother as described in detail herein.

The term “immunosuppressive agent” as used herein for adjunct therapyrefers to substances that act to suppress or mask the immune system ofthe mammal being treated herein. This would include substances thatsuppress cytokine production, down-regulate or suppress self-antigenexpression, or mask the MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077);nonsteroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus,glucocorticoids such as cortisol or aldosterone, anti-inflammatoryagents such as a cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor,or a leukotriene receptor antagonist; purine antagonists such asazathioprine or mycophenolate mofetil (MMF); alkylating agents such ascyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (whichmasks the MHC antigens, as described in U.S. Pat. No. 4,120,649);anti-idiotypic antibodies for MEC antigens and MEC fragments;cyclosporin A; steroids such as corticosteroids or glucocorticosteroidsor glucocorticoid analogs, e.g., prednisone, methylprednisolone, anddexamethasone; dihydrofolate reductase inhibitors such as methotrexate(oral or subcutaneous); hydroxycloroquine; sulfasalazine; leflunomide;cytokine or cytokine receptor antagonists includinganti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosisfactor-alpha antibodies (infliximab or adalimumab), anti-TNF-alphaimmunoahesin (etanercept), anti-tumor necrosis factor-beta antibodies,anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies;anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies;anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-Tantibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; solublepeptide containing a LFA-3 binding domain (WO 90/08187 published Jul.26, 1990); streptokinase; TGF-beta; streptodornase; RNA or DNA from thehost; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor(Cohen et al., U.S. Pat. No. 5,114,721); T-cell receptor fragments(Offner et al, Science, 251: 430-432 (1991); WO 90/11294; Ianeway,Nature, 341: 482 (1989); and WO 91/01133); and T cell receptorantibodies (EP 340,109) such as T10B9.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegall (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANECremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those thatinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;vaccines such as gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2;LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

The term “cytokine” is a generic term for proteins released by one cellpopulation that act on another cell as intercellular mediators. Examplesof such cytokines are lymphokines, monokines; interleukins (ILs) such asIL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11,IL-12, IL-15; a tumor necrosis factor such as TNF-α or TNF-β; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines, including synthetically producedsmall-molecule entities and pharmaceutically acceptable derivatives andsalts thereof.

The term “hormone” refers to polypeptide hormones, which are generallysecreted by glandular organs with ducts. Included among the hormonesare, for example, growth hormone such as human growth hormone,N-methionyl human growth hormone, and bovine growth hormone; parathyroidhormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;glycoprotein hormones such as follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), and luteinizing hormone (LH);prolactin, placental lactogen, mouse gonadotropin-associated peptide,inhibin; activin; mullerian-inhibiting substance; and thrombopoietin. Asused herein, the term hormone includes proteins from natural sources orfrom recombinant cell culture and biologically active equivalents of thenative sequence hormone, including synthetically produced small-moleculeentities and pharmaceutically acceptable derivatives and salts thereof.

The term “growth factor” refers to proteins that promote growth, andinclude, for example, hepatic growth factor; fibroblast growth factor;vascular endothelial growth factor; nerve growth factors such as NGF-β;platelet-derived growth factor; transforming growth factors (TGFs) suchas TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin(EPO); osteoinductive factors; interferons such as interferon-α, -β, and-γ; and colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF). As used herein, the term growth factor includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence growth factor, includingsynthetically produced small-molecule entities and pharmaceuticallyacceptable derivatives and salts thereof.

The term “integrin” refers to a receptor protein that allows cells bothto bind to and to respond to the extracellular matrix and is involved ina variety of cellular functions such as wound healing, celldifferentiation, homing of tumor cells and apoptosis. They are part of alarge family of cell adhesion receptors that are involved incell-extracellular matrix and cell-cell interactions. Functionalintegrins consist of two transmembrane glycoprotein subunits, calledalpha and beta that are non-covalently bound. The alpha subunits allshare some homology to each other, as do the beta subunits. Thereceptors always contain one alpha chain and one beta chain. Examplesinclude Alpha6beta1, Alpha3beta1, Alpha7beta1, LFA-1, alpha 4 integrinetc. As used herein, the term integrin includes proteins from naturalsources or from recombinant cell culture and biologically activeequivalents of the native sequence integrin, including syntheticallyproduced small-molecule entities and pharmaceutically acceptablederivatives and salts thereof.

Examples of “integrin antagonists or antibodies” herein include an LFA-1antibody such as Efalizumab (RAPTIVA®) commercially available fromGenentech; an alpha 4 integrin antibody such as natalizumab (TYSABRI®)available from Biogen Idec/Elan Pharmaceuticals, Inc.; diazacyclicphenylalanine derivatives (WO 2003/89410); phenylalanine derivatives (WO2003/70709, WO 2002/28830, WO 2002/16329 and WO 2003/53926);phenylpropionic acid derivatives (WO 2003/10135); enamine derivatives(WO 2001/79173); propanoic acid derivatives (WO 2000/37444); alkanoicacid derivatives (WO 2000/32575); substituted phenyl derivatives (U.S.Pat. Nos. 6,677,339 and 6,348,463); aromatic amine derivatives (U.S.Pat. No. 6,369,229); and ADAM disintegrin domain polypeptide(US2002/0042368), antibodies to alphavbeta3 integrin (EP 633945);aza-bridged bicyclic amino acid derivatives (WO 2002/02556) etc.

For the purposes herein, “tumor necrosis factor alpha (TNF-alpha)”refers to a human TNF-alpha molecule comprising the amino acid sequenceas described in Pennica et al., Nature, 312:721 (1984) or Aggarwal etal., JBC, 260:2345 (1985).

A “TNF-alpha inhibitor” herein is an agent that inhibits, to someextent, a biological function of TNF-alpha, generally through binding toTNF-alpha and neutralizing its activity. Examples of TNF inhibitorsspecifically contemplated herein are Etanercept (ENBREL®), Infliximab(REMICADE®) and Adalimumab (HUMMIRA™).

Examples of “disease-modifying anti-rheumatic drugs” or “DMARDs” includehydroxycloroquine, sulfasalazine, methotrexate, leflunomide, etanercept,infliximab (plus oral and subcutaneous methrotrexate), azathioprine,D-penicillamine, Gold (oral), Gold (intramuscular), minocycline,cyclosporine, Staphylococcal protein A immunoadsorption, including saltsand derivatives thereof, etc.

Examples of “nonsteroidal anti-inflammatory drugs” or “NSAIDs” areacetylsalicylic acid, ibuprofen, naproxen, indomethacin, sulindac,tolmetin, including salts and derivatives thereof, etc.

“Corticosteroid” refers to any one of several synthetic or naturallyoccurring substances with the general chemical structure of steroidsthat mimic or augment the effects of the naturally occurringcorticosteroids. Examples of synthetic corticosteroids includeprednisone, prednisolone (including methylprednisolone), dexamethasone,glucocorticoid and betamethasone.

A “package insert” is used to refer to instructions customarily includedin commercial packages of therapeutic products, that contain informationabout the indications, usage, dosage, administration, contraindications,other therapeutic products to be combined with the packaged product,and/or warnings concerning the use of such therapeutic products, etc.

A “label” is used herein to refer to information customarily includedwith commercial packages of pharmaceutical formulations includingcontainers such as vials and package inserts, as well as other types ofpackaging.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.′

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that aspects and variations of the inventiondescribed herein include “consisting” and/or “consisting essentially of”aspects and variations.

II. Methods of Treatment

The present invention provides methods of treating progressive multiplesclerosis in a patient comprising administering to the patient aneffective amount of an anti-CD20 antibody.

In some embodiments, the invention provides methods of treatingprogressive multiple sclerosis in a patient comprising administering tothe patient an effective amount of an anti-CD20 antibody, whereintreatment is based upon the patient having one or more characteristicsselected from the group consisting of (a) an age less than about 55years, (b) one or more gadolinium staining lesions, (c) at least about aone point increase in Expanded Disability Status Scale (EDSS) over twoyears prior to starting treatment, and (d) a Multiple Sclerosis SeverityScore (MSSS) greater than about 5 points.

In some embodiments, the invention provides methods of treatingprogressive multiple sclerosis in a patient provided that the patienthas been found to have one or more characteristics selected from thegroup consisting of (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, (c) at least about a one point increase inExpanded Disability Status Scale (EDSS) over two years prior to startingthe treatment, and (d) a Multiple Sclerosis Severity Score (MSSS)greater than about 5 points, the treatment comprising administering tothe patient an effective amount of an anti-CD20 antibody.

In some embodiments, the invention provides methods of treatingprogressive multiple sclerosis in a patient comprising administering tothe patient an effective amount of an anti-CD20 antibody, wherein thepatient has at the time of starting treatment one or morecharacteristics selected from the group consisting of (a) an age lessthan about 55 years, (b) one or more gadolinium staining lesions, (c) atleast about a one point increase in Expanded Disability Status Scale(EDSS) over two years prior to starting treatment, and (d) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points, wherebyevidence of the age, the gadolinium staining lesions, the increase inEDDS over two years prior to starting the treatment, MSSS, or acombination thereof indicates that the patient will respond to treatmentwith the anti-CD20 antibody.

In some embodiments, the invention provides methods of treatingprogressive multiple sclerosis, comprising: (a) selecting a patient hasone or more characteristics selected from the group consisting of (i) anage less than about 55 years, (ii) one or more gadolinium staininglesions, (iii) at least about a one point increase in ExpandedDisability Status Scale (EDSS) over two years prior to startingtreatment, and (iv) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points; and (b) administering to the patient thus selectedan effective amount of an anti-CD20 antibody.

In some embodiments, the invention provides methods of treatingprogressive multiple sclerosis in a patient, comprising administering tothe patient an effective amount of an anti-CD20 antibody, and whereinone or more characteristics selected from the group consisting of (a) anage less than about 55 years, (b) one or more gadolinium staininglesions, (c) at least about a one point increase in Expanded DisabilityStatus Scale (EDSS) over two years prior to starting treatment, and (d)a Multiple Sclerosis Severity Score (MSSS) greater than about 5points.is used as a basis for selecting the patient to receive thetreatment, and wherein said treatment comprises administering aneffective amount of the anti-CD20 antibody to the patient.

The invention further provides methods of treating multiple sclerosis ina patient comprising administering an effective amount of ocrelizumab tothe patient to provide an initial ocrelizumab exposure of between about0.3 to about 0.6 grams followed by a second ocrelizumab exposure ofbetween about 0.3 to about 0.6 grams, the second exposure not beingprovided until from about 16 to 60 weeks from the initial exposure, andeach of the ocrelizumab exposures is provided to the patient as one ortwo doses of ocrelizumab. In some embodiments, the initial ocrelizumabexposure is about 0.6 grams. In some embodiments, the second ocrelizumabexposure is about 0.6 grams. In some embodiments, the second exposure isadministered from about 24 weeks from the initial exposure. In someembodiments, one or more of the ocrelizumab exposures are provided tothe patient as one dose of ocrelizumab. In some embodiments, one or moreof the ocrelizumab exposures are provided to the patient as two doses ofocrelizumab. In some embodiments, the two doses of ocrelizumab compriseabout 0.3 grams of ocrelizumab.

The present invention also provides methods of assessing and/orpredicting responsiveness of a patient with progressive multiplesclerosis to an anti-CD20 antibody treatment.

In some embodiments, the invention provides methods of assessing whethera patient with progressive multiple sclerosis will respond to treatmentwith an anti-CD20 antibody comprising assessing one or morecharacteristics selected from the group consisting of (a) an age lessthan about 55 years, (b) one or more gadolinium staining lesions, (c) atleast about a one point increase in Expanded Disability Status Scale(EDSS) over two years prior to starting treatment, and (d) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points, wherein oneor more of the characteristics in the patient indicates the patient willbe responsive to the treatment.

In some embodiments, the invention provides methods of assessing whethera patient with progressive multiple sclerosis will respond to treatmentwith an anti-CD20 antibody comprising: (a) assessing one or morecharacteristics selected from the group consisting of (i) an age lessthan about 55 years, (ii) one or more gadolinium staining lesions, (iii)at least about a one point increase in Expanded Disability Status Scale(EDSS) over two years prior to starting treatment, and (iv) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points; (b)implementing an algorithm to determine that the patient is responsive tosaid treatment; and (c) recording a result specific to the patient beingtested.

In some embodiments, the invention provides methods of treatingprogressive multiple sclerosis in a patient comprising: (a) assessingone or more characteristics selected from the group consisting of (i) anage less than about 55 years, (i) one or more gadolinium staininglesions, (iii) at least about a one point increase in ExpandedDisability Status Scale (EDSS) over two years prior to startingtreatment, and (iv) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points, wherein the patient is selected for treatment basedon the patient having one or more characteristics selected from thegroup consisting of (i) an age less than about 55 years, (ii) one ormore gadolinium staining lesions, (iii) at least about a one pointincrease in Expanded Disability Status Scale (EDSS) over two years priorto starting treatment, and (iv) a Multiple Sclerosis Severity Score(MSSS) greater than about 5 points; and (b) treating said selectedpatient by administering to said selected patient an effective amount ofan anti-CD20 antibody.

In some embodiments, the invention provides methods for selecting atherapy for a patient and/or a patient population with progressivemultiple sclerosis comprising: (a) assessing one or more characteristicsselected from the group consisting of (i) an age less than about 55years, (ii) one or more gadolinium staining lesions, (iii) at leastabout a one point increase in Expanded Disability Status Scale (EDSS)over two years prior to starting treatment, and (iv) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points; and (b)selecting an anti-CD20 antibody for treatment if the patient or patientpopulation has one or more characteristics selected from the groupconsisting of (i) an age less than about 55 years, (ii) one or moregadolinium staining lesions, (iii) at least about a one point increasein Expanded Disability Status Scale (EDSS) over two years prior tostarting the anti-CD20 treatment, and (iv) a Multiple Sclerosis SeverityScore (MSSS) greater than about 5 points.

In some embodiments, the invention provides methods for predictingwhether a patient with progressive multiple sclerosis will respond to ananti-CD20 antibody, the methods comprising assessing one or morecharacteristics selected from the group consisting of (a) an age lessthan about 55 years, (b) one or more gadolinium staining lesions, (c) atleast about a one point increase in Expanded Disability Status Scale(EDSS) over two years prior to starting treatment, and (d) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points, whereby theage, the gadolinium staining lesions, the increase in EDDS over twoyears prior to starting treatment, MSSS, or a combination thereofindicates that the patient will respond to the anti-CD20 antibody.

In some embodiments, the invention provides methods of identifying apatient with progressive multiple sclerosis likely to respond toanti-CD20 antibody treatment comprising: (a) assessing one or morecharacteristics selected from the group consisting of (i) an age lessthan about 55 years, (ii) one or more gadolinium staining lesions, (iii)at least about a one point increase in Expanded Disability Status Scale(EDSS) over two years prior to starting treatment, and (iv) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points; and (b)identifying the patient having one or more characteristics selected fromthe group consisting of (i) an age less than about 55 years, (ii) one ormore gadolinium staining lesions, (iii) at least about a one pointincrease in Expanded Disability Status Scale (EDSS) over two years priorto starting treatment, and (iv) a Multiple Sclerosis Severity Score(MSSS) greater than about 5 points.

In some embodiments of any of the methods of treating, assessing, and/orpredicting responsiveness described herein, the patients have one ormore characteristics selected from the group consisting of (a) an ageless than about 55 years, (b) one or more gadolinium staining lesions,and (c) at least about a one point increase in Expanded DisabilityStatus Scale (EDSS) over two years prior to starting treatment.

In some embodiments of any of the methods of assessing and/or predictingresponsiveness described herein, the methods further comprise advising apatient.

In some embodiments of any of the methods of treating and assessingand/or predicting responsiveness described herein, the patient has morethan one characteristics selected from the group consisting of (a) anage less than about 55 years, (b) one or more gadolinium staininglesions, (c) at least about a one point increase in Expanded DisabilityStatus Scale (EDSS) over two years prior to starting treatment, and (d)a Multiple Sclerosis Severity Score (MSSS) greater than about 5 points.In some embodiments of any of the methods of treating and assessingand/or predicting responsiveness described herein, the patient has twocharacteristics selected from the group consisting of (a) an age lessthan about 55 years, (b) one or more gadolinium staining lesions, (c) atleast about a one point increase in Expanded Disability Status Scale(EDSS) over two years prior to starting treatment, and (d) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points. In someembodiments, the patient has three characteristics selected from thegroup consisting of (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, and (c) at least about a one point increasein EDSS over two years prior to starting treatment. In some embodiments,the patient has (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, (c) at least about a one point increase inExpanded Disability Status Scale (EDSS) over two years prior to startingtreatment, and (d) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points.

In some embodiments of any of the methods of treating and assessingand/or predicting responsiveness described herein, the progressivemultiple sclerosis is primary progressive multiple sclerosis. In someembodiments, the progressive multiple sclerosis is secondary progressivemultiple sclerosis. In some embodiments, the progressive multiplesclerosis is progressive relapsing multiple sclerosis. In someembodiments, the patient and/or patient population is not diagnosed withrelapsing remitting multiple sclerosis when starting treatment.

In some embodiments of any of the methods of treating and assessingand/or predicting responsiveness described herein, the patient and/orpatient population further has evidence of inflammation in a sample.Evidence of inflammation is indicated by assessing one or more indiciaof inflammation. The sample can be any suitable sample to assessinflammation. In some embodiments, the sample is tissue or fluid. Insome embodiments, the fluid sample is a cerebrospinal fluid sample. Insome embodiments, the evidence of inflammation is indicated by anelevated IgG index. In some embodiments, the evidence of inflammation isindicated by IgG oligoclonal bands detected by isoelectric focusing. Insome embodiments, the evidence of inflammation is detected by MRI. Insome embodiments, the evidence of inflammation is detected by thepresence of Gd-enhancing lesions or T2 lesions. Other methods ofassessing evidence of inflammation are known in the art.

In some embodiments of any of the methods of treating and assessingand/or predicting responsiveness described herein, the patient and/orpatient population is characterized by a change in EDSS over a period oftime. In some embodiments, the patient and/or patient population ischaracterized by at least about any of 1 point, 1.25 point, 1.5 point,1.75 point, 2 point, 2.25 point, 2.5 point, 2.75 point, or 3 pointincrease in EDSS over two years prior to starting the anti-CD20 antibodytreatment. In some embodiments, the increase in EDSS is at least about a1.5 point increase in EDSS over two years prior to starting treatment.In some embodiments, the increase in EDSS over two years prior tostarting treatment is not attributable to relapse. In some embodiments,the patient and/or patient population is characterized by having had anEDSS of greater than about 5.0 for less than about any of 5 years, 6years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, or 20years. In some embodiments, the patient and/or patient population ischaracterized by having had an EDSS of greater than about 5.0 for lessthan about 15 years. In some embodiments, the patient and/or patientpopulation is characterized by having had an EDSS less than or equal toabout 5.0 for less than about any of 5 years, 6 years, 7 years, 8 years,9 years, 10 years, 11 years, 12 years, 13 years, 14 years, or 15 years.In some embodiments, the patient and/or patient population ischaracterized by having had an EDSS less than or equal to about 5.0 forless than about 10 years.

In some embodiments of any of the methods of treating and assessingand/or predicting responsiveness described herein, the patient and/orpatient population is characterized by having an EDSS of between aboutany of 1.5 points to 7 points, 1.5 points to 6.5 points, 2 points to 6.5points, or 3 points to 6.5 points. In some embodiments, the patientand/or patient population is characterized by having an EDSS whenstarting treatment is between about 3.0 and about 6.5.

In some embodiments of any of the methods of treating, assessing and/orpredicting responsiveness described herein, the patient and/or patientpopulation is characterized by having an MSSS of greater than about anyof 6, 7, 8, or 9. In some embodiments of any of the methods of treatingand assessing and/or predicting responsiveness described herein, thepatient and/or patient population is characterized by having an MSSS ofgreater than about 9.

In some embodiments of any of the methods of treating and assessingand/or predicting responsiveness described herein, the patient and/orpatient population is further characterized by having two or morerelapses within two years prior to starting treatment. In someembodiments, the patient and/or patient population is furthercharacterized by having any of 2 relapses, 3 relapses, 4 relapses, or 5relapses within two years prior to starting treatment.

In some embodiments of any of the methods of treating and assessingand/or predicting responsiveness described herein, the patient and/orpatient population is characterized by an age. In some embodiments, thepatient and/or patient population is characterized by having an age lessthan about any of 55 years, 54 years, 53 years, 52 years, 51 years, or50 years. In some embodiments, the patient and/or patient population ischaracterized by having an age of less than about 51 years.

In some embodiments of any of the methods of treating and assessingand/or predicting responsiveness described herein, the treatment reducestime to confirmed disease progression. In some embodiments, theconfirmed disease progression is an increase in EDSS that is sustainedfor about any of 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24weeks, 28 weeks, or 32 weeks. In some embodiments, the confirmed diseaseprogression is an increase in EDSS that is sustained for twelve weeks.In some embodiments, the confirmed disease progression is an increase inEDSS that is sustained for twenty-four weeks.

In some embodiments of any of the methods of treating and assessingand/or predicting responsiveness described herein, the anti-CD20antibody comprises: a) a heavy chain variable region comprising SEQ IDNO:10, SEQ ID NO:11, and SEQ ID NO:12, and b) a light chain variableregion comprising SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In someembodiments of any of the methods described herein, the anti-CD20antibody is ocrelizumab. In some embodiments of any of the methodsdescribed herein, the anti-CD20 antibody is rituximab. In someembodiments of any of the methods described herein, the anti-CD20antibody is ofatumumab. In some embodiments of any of the methodsdescribed herein, the anti-CD20 antibody is TRU-015 or SBI-087. In someembodiments of any of the methods described herein, the anti-CD20antibody is GA101. In some embodiments of any of the methods describedherein, the anti-CD20 antibody is hA20.

The methods described herein may encompass any combination of theembodiments described herein. For example, the methods include methodsof treatment and assessing and/or predicting wherein the patient has (a)an age less than about 55 years and (b) one or more gadolinium staininglesions.

III. Dosages

According to some embodiments of any of the methods or articles ofmanufacture described herein, the method or instructions comprisesadministering an effective amount of an anti-CD20 antibody to themultiple sclerosis patient to provide an initial antibody exposure ofabout 0.3 to about 4 grams (preferably about 0.3 to about 1.5 grams,such as about 0.6 grams or about 1.0 grams) followed by a secondantibody exposure of about 0.3 to about 4 grams (preferably about 0.3 toabout 1.5 grams, such as about 0.6 grams or about 1.0 grams), the secondantibody exposure not being provided until from about 16 to about 60weeks from the initial antibody exposure. For purposes of thisinvention, the second antibody exposure is the next time the patient istreated with the anti-CD20 antibody after the initial antibody exposure,there being no intervening anti-CD20 antibody treatment or exposurebetween the initial and second exposures. In some embodiments, theinitial antibody exposure and/or the second antibody exposure is aboutany of 0.3 grams, 0.4 grams, 0.5 grams, 0.6 grams, 0.7 grams, 0.8 grams,0.9 grams, or 1.0 grams.

The interval between the initial and second or subsequent antibodyexposures can be measured from either the first or second dose of theinitial antibody exposure, but in some embodiments, from the first doseof the initial antibody exposure.

In some embodiments, the antibody exposures are approximately 24 weeksor 6 months apart; or approximately 48 weeks or 12 months apart.

In one embodiment, the second antibody exposure is not provided untilabout 20 to about 30 weeks from the initial exposure, optionallyfollowed by a third antibody exposure of about 0.3 to about 4 grams(preferably about 0.3 to about 1.5 grams), the third exposure not beingadministered until from about 46 to 60 weeks (preferably from about 46to 54 weeks) from the initial exposure, and then, in some embodiments,no further antibody exposure is provided until at least about 70-75weeks from the initial exposure. In some embodiments, the third antibodyexposure is about any of 0.3 grams, 0.4 grams, 0.5 grams, 0.6 grams, 0.7grams, 0.8 grams, 0.9 grams, or 1.0 grams.

In an alternative embodiment, the second antibody exposure is notprovided until about 46 to 60 weeks from the initial exposure, andsubsequent antibody exposures, if any, are not provided until about 46to 60 weeks from the previous antibody exposure.

Any one or more of the antibody exposures herein may be provided to thepatient as a single dose of antibody, or as two separate doses of theantibody (i.e., constituting a first and second dose). The particularnumber of doses (whether one or two) employed for each antibody exposureis dependent, for example, on the type of MS treated, the type ofantibody employed, whether and what type of second medicament isemployed, and the method and frequency of administration. Where twoseparate doses are administered, the second dose is preferablyadministered from about 3 to 17 days, more preferably from about 6 to 16days, and most preferably from about 13 to 16 days from the time thefirst dose was administered. In some embodiments, where two separatedoses are administered, the second dose is about 14 days. Where twoseparate doses are administered, the first and second dose of theantibody is preferably about 0.3 to 1.5 grams, more preferably about 0.3to about 1.0 grams. In some embodiments, where two separate doses areadministered, the first and second dose of the antibody is about any of0.3 grams, 0.4 grams, 0.5 grams, or 0.6 grams. In some embodiments, theinitial ocrelizumab exposure comprises a first dose and a second dose ofocrelizumab, wherein the first dose and second dose of ocrelizumab isabout 0.3 grams. In some embodiments, the second ocrelizumab exposurecomprises a single dose of ocrelizumab, wherein the single dose ofocrelizumab is 0.6 grams.

In one embodiment, the patient is provided at least about three, atleast about four, or at least about five exposures of the antibody, forexample, from about 3 to 60 exposures, and more particularly about 3 to40 exposures, most particularly, about 3 to 20 exposures. In someembodiments of any of the methods, the methods further comprisingproviding between about one to about three subsequent ocrelizumabexposures. In some embodiments, such exposures are administered atintervals each of approximately 24 weeks or 6 months, or 48 weeks or 12months. In one embodiment, each antibody exposure is provided as asingle dose of the antibody. In an alternative embodiment, each antibodyexposure is provided as two separate doses of the antibody. However, notevery antibody exposure need be provided as a single dose or as twoseparate doses.

The antibody may be a naked antibody or may be conjugated with anothermolecule such as a cytotoxic agent such as a radioactive compound. Insome embodiments, the antibody is Rituximab, humanized 2H7 (e.g.comprising the variable domain sequences in SEQ ID NOS. 2 and 8) orhumanized 2H7 comprising the variable domain sequences in SEQ ID NOS. 23and 24, or huMax-CD20 (Genmab). In some embodiments, the antibody isocrelizumab (e.g., comprising (a) a light chain comprising the aminoacid sequence of SEQ ID NO: 13 and (b) a heavy chain comprising theamino acid sequence of SEQ ID NO:14).

In one embodiment, the patient has never been previously treated withdrug(s), such as immunosuppressive agent(s), to treat the multiplesclerosis and/or has never been previously treated with an antibody to aB-cell surface marker (e.g. never previously treated with a CD20antibody).

The antibody is administered by any suitable means, includingparenteral, topical, subcutaneous, intraperitoneal, intrapulmonary,intranasal, and/or intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Intrathecal administration is alsocontemplated (see, e.g., US Patent Appln No. 2002/0009444, Grillo-Lopez,A concerning intrathecal delivery of a CD20 antibody). In addition, theantibody may suitably be administered by pulse infusion, e.g., withdeclining doses of the antibody. In some embodiments, the dosing isgiven intravenously, subcutaneously or intrathecally. In someembodiments, the dosing is given by intravenous infusion(s).

While the CD20 antibody may be the only drug administered to the patientto treat the multiple sclerosis, one may optionally administer a secondmedicament, such as a cytotoxic agent, chemotherapeutic agent,immunosuppressive agent, cytokine, cytokine antagonist or antibody,growth factor, hormone, integrin, integrin antagonist or antibody (e.g.an LFA-1 antibody such as efalizumab (RAPTIVA®) commercially availablefrom Genentech, or an alpha 4 integrin antibody such as natalizumab(TYSABRI®) available from Biogen Idec/Elan Pharmaceuticals, Inc) etc,with the antibody that binds a B cell surface marker (e.g. with the CD20antibody).

In some embodiments of combination therapy, the antibody is combinedwith an interferon class drug such as IFN-beta-la (REBIF® and AVONEX®)or IFN-beta-lb (BETASERON®); an oligopeptide such a glatiramer acetate(COPAXONE®); a cytotoxic agent such as mitoxantrone (NOVANTRONE®),methotrexate, cyclophosphamide, chlorambucil, azathioprine; intravenousimmunoglobulin (gamma globulin); lymphocyte-depleting therapy (e.g.,mitoxantrone, cyclophosphamide, Campath, anti-CD4, cladribine, totalbody irradiation, bone marrow transplantation); corticosteroid (e.g.methylprednisolone, prednisone, dexamethasone, or glucorticoid),including systemic corticosteroid therapy; non-lymphocyte-depletingimmunosuppressive therapy (e.g., mycophenolate mofetil (MMF) orcyclosporine); cholesterol-lowering drug of the “statin” class, whichincludes cerivastatin (BAYCOL®), fluvastatin (LESCOL®), atorvastatin(LIPITOR®), lovastatin (MEVACOR®), pravastatin (PRAVACHOL®), Simvastatin(ZOCOR®); estradiol; testosterone (optionally at elevated dosages; Stuveet al. Neurology 8:290-301 (2002)); hormone replacement therapy;treatment for symptoms secondary or related to MS (e.g., spasticity,incontinence, pain, fatigue); a TNF inhibitor; disease-modifyinganti-rheumatic drug (DMARD); non-steroidal anti-inflammatory drug(NSAID); plasmapheresis; levothyroxine; cyclosporin A; somatastatinanalogue; cytokine or cytokine receptor antagonist; anti-metabolite;immunosuppressive agent; rehabilitative surgery; radioiodine;thyroidectomy; another B-cell surface antagonist/antibody; etc.

The second medicament is administered with the initial exposure and/orlater exposures of the CD20 antibody, such combined administrationincludes co-administration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities.

Aside from administration of antibodies to the patient, the presentapplication contemplates administration of antibodies by gene therapy.Such administration of nucleic acid encoding the antibody is encompassedby the expression administering an “effective amount” of an antibody.See, for example, WO96/07321 published Mar. 14, 1996 concerning the useof gene therapy to generate intracellular antibodies.

There are two major approaches to getting the nucleic acid (optionallycontained in a vector) into the patient's cells; in vivo and ex vivo.For in vivo delivery the nucleic acid is injected directly into thepatient, usually at the site where the antibody is required. For ex vivotreatment, the patient's cells are removed, the nucleic acid isintroduced into these isolated cells and the modified cells areadministered to the patient either directly or, for example,encapsulated within porous membranes that are implanted into the patient(see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187). There are a varietyof techniques available for introducing nucleic acids into viable cells.The techniques vary depending upon whether the nucleic acid istransferred into cultured cells in vitro, or in vivo in the cells of theintended host. Techniques suitable for the transfer of nucleic acid intomammalian cells in vitro include the use of liposomes, electroporation,microinjection, cell fusion, DEAE-dextran, the calcium phosphateprecipitation method, etc. A commonly used vector for ex vivo deliveryof the gene is a retrovirus.

In some embodiments, the in vivo nucleic acid transfer techniquesinclude transfection with viral vectors (such as adenovirus, Herpessimplex I virus, or adeno-associated virus) and lipid-based systems(useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPEand DC-Chol, for example). In some situations it is desirable to providethe nucleic acid source with an agent that targets the target cells,such as an antibody specific for a cell surface membrane protein or thetarget cell, a ligand for a receptor on the target cell, etc. Whereliposomes are employed, proteins that bind to a cell surface membraneprotein associated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins that undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87:3410-3414 (1990). For review of the currently knowngene marking and gene therapy protocols see Anderson et al., Science256:808-813 (1992). See also WO 93/25673 and the references citedtherein.

IV. Antibodies and their Production

The methods and articles of manufacture of the present invention use, orincorporate, an antibody that binds to a B-cell surface marker,especially one that binds to CD20. Accordingly, methods for generatingsuch antibodies will be described here.

The B cell surface marker to be used for production of, or screeningfor, antibodies may be, e.g., a soluble form of the marker or a portionthereof, containing the desired epitope. Alternatively, or additionally,cells expressing the marker at their cell surface can be used togenerate, or screen for, antibodies. Other forms of the B cell surfacemarker useful for generating antibodies will be apparent to thoseskilled in the art.

A description follows as to exemplary techniques for the production ofthe antibodies used in accordance with the present invention.

(i) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R′N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. In some embodiments, the animal isboosted with the conjugate of the same antigen, but conjugated to adifferent protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

(ii) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical and/or bind the same epitope except forpossible variants that arise during production of the monoclonalantibody, such variants generally being present in minor amounts. Thus,the modifier “monoclonal” indicates the character of the antibody as notbeing a mixture of discrete or polyclonal antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as herein described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

In some embodiments, the myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these, in some embodiments, the myeloma cell lines aremurine myeloma lines, such as those derived from MOPC-21 and MPC-11mouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen. Insome embodiments, the binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by immunoprecipitation or byan in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al, Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). In some embodiments, the hybridomacells serve as a source of such DNA. Once isolated, the DNA may beplaced into expression vectors, which are then transfected into hostcells such as E. coli cells, simian COS cells, Chinese Hamster Ovary(CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. Review articles on recombinant expressionin bacteria of DNA encoding the antibody include Skerra et al., Curr.Opinion in Immunol, 5:256-262 (1993) and Plückthun, Immunol. Revs.,130:151-188 (1992).

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iii) Humanized Antibodies

Methods for humanizing non-human antibodies have been described in theart. In some embodiments, a humanized antibody has one or more aminoacid residues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann etal, Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536(1988)), by substituting hypervariable region sequences for thecorresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence that is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al, J. Mol. Biol., 196:901(1987)). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chain variable regions. The same framework may be usedfor several different humanized antibodies (Carter et al., Proc. Natl.Acad. Sci. USA, 89:4285 (1992); Presta et al, J. Immunol., 151:2623(1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, in some embodiments of the methods, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablethat illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

In some embodiments, the humanized anti-CD20 antibody is a humanized 2H7antibody. In some embodiments, the humanized 2H7 antibody preferablycomprises one, two, three, four, five or six of the following CDRsequences:

-   -   CDR L1 sequence RASSSVSYXH wherein X is M or L (SEQ ID NO. 18),        for example SEQ ID NO:4 (FIG. 1A),    -   CDR L2 sequence of SEQ ID NO:5 (FIG. 1A),    -   CDR L3 sequence QQWXFNPPT wherein X is S or A (SEQ ID NO. 19),        for example SEQ ID NO:6 (FIG. 1A),    -   CDR H1 sequence of SEQ ID NO:10 (FIG. 1B),    -   CDR H2 sequence of AIYPGNGXTSYNQKFKG wherein X is D or A (SEQ ID        NO. 20), for example SEQ ID NO:11 (FIG. 1B), and    -   CDR H3 sequence of VVYYSXXYWYFDV wherein the X at position 6 is        N, A, Y, W or D, and the X as position 7 is S or R (SEQ ID NO.        21), for example SEQ ID NO:12 (FIG. 1B).

The CDR sequences above are generally present within human variablelight and variable heavy framework sequences, such as substantially thehuman consensus FR residues of human light chain kappa subgroup I(V_(L)6I), and substantially the human consensus FR residues of humanheavy chain subgroup III (V_(H)III). See also WO 2004/056312 (Lowman etal.).

In some embodiments, the variable heavy region may be joined to a humanIgG chain constant region, wherein the region may be, for example, IgG1or IgG3, including native sequence and variant constant regions.

In some embodiments, such antibody comprises the variable heavy domainsequence of SEQ ID NO:8 (v16, as shown in FIG. 1B), optionally alsocomprising the variable light domain sequence of SEQ ID NO:2 (v16, asshown in FIG. 1A), which optionally comprises one or more amino acidsubstitution(s) at positions 56, 100, and/or 100a, e.g. D56A, N100A orN100Y, and/or S100aR in the variable heavy domain and one or more aminoacid substitution(s) at positions 32 and/or 92, e.g. M32L and/or S92A,in the variable light domain. In some embodiments, the antibody is anintact antibody comprising the light chain amino acid sequences of SEQID NOs. 13 or 16, and heavy chain amino acid sequences of SEQ ID NO. 14,15, 17, 22 or 25. In some embodiments, the humanized 2H7 antibody isocrelizumab (Genentech).

In the embodiments, the humanized 2H7 is an intact antibody or antibodyfragment comprising the variable light chain sequence:

(SEQ ID NO: 2) DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQW SFNPPTFGQGTKVEIKR;and the variable heavy chain sequence: (SEQ ID NO: 8)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS.

In some embodiments, the humanized 2H7 antibody is an intact antibody,in some embodiments, it comprises the light chain amino acid sequence:

(SEQ ID NO: 13) DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;and the heavy chain amino acid sequence: (SEQ ID NO: 14)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMEIWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK or the heavy chain amino acid sequence: (SEQ ID NO: 15)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMEIWVRQAPGKGLEWVGAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK.

In some embodiments, the humanized 2H7 antibody comprises 2H7.v511variable light domain sequence:

(SEQ ID NO: 23) DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ  QWAFNPPTFGQGTKVEIKRand 2H7.v511 variable heavy domain sequence: (SEQ ID NO. 24)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMEWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSS.

In some embodiments, the humanized 2H7.v511 antibody is an intactantibody, it may comprise the light chain amino acid sequence:

(SEQ ID NO: 16) DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC and the heavy chain amino acid sequence of  SEQ ID NO. 17 or:(SEQ ID NO. 25) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMEIWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVSC SVMHEALHNHYTQKSLSLSPG.

In some embodiments, the antibody herein may further comprise at leastone amino acid substitution in the Fc region that improves ADCCactivity, such as one wherein the amino acid substitutions are atpositions 298, 333, and 334, preferably S298A, E333A, and K334A, usingEu numbering of heavy chain residues. See also U.S. Pat. No.6,737,056B1, Presta. Any of these antibodies may comprise at least onesubstitution in the Fc region that improves FcRn binding or serumhalf-life, for example a substitution at heavy chain position 434, suchas N434W. See also U.S. Pat. No. 6,737,056B1, Presta. Any of theseantibodies may further comprise at least one amino acid substitution inthe Fc region that increases CDC activity, for example, comprising atleast a substitution at position 326, preferably K326A or K326W. Seealso U.S. Pat. No. 6,528,624B1 (Idusogie et al.).

In some embodiments, the humanized 2H7 variants are those comprising thevariable light domain of SEQ ID NO:2 and the variable heavy domain ofSEQ ID NO:8, including those with or without substitutions in an Fcregion (if present), and those comprising a variable heavy domain withalteration N100A; or D56A and N100A; or D56A, N100Y, and S100aR; in SEQID NO:8 and a variable light domain with alteration M32L; or S92A; orM32L and S92A; in SEQ ID NO:2. M34 in the variable heavy domain of2H7.v16 has been identified as a potential source of antibody stabilityand is another potential candidate for substitution.

In some embodiments of the invention, the variable region of variantsbased on 2H7.v16 comprise the amino acid sequences of v16 except at thepositions of amino acid substitutions that are indicated in the tablebelow. Unless otherwise indicated, the 2H7 variants will have the samelight chain as that of v16.

TABLE 1 Exemplary Humanized 2H7 Antibody Variants 2H7 Heavy chain Lightchain Version (V_(H)) changes (V_(L)) changes Fc changes 16 forreference — 31 — — S298A, E333A, K334A 73 N100A M32L 75 N100A M32LS298A, E333A, K334A 96 D56A, N100A S92A 114 D56A, N100A M32L, S92AS298A, E333A, K334A 115 D56A, N100A M32L, S92A S298A, E333A, K334A,E356D, M358L 116 D56A, N100A M32L, S92A S298A, K334A, K322A 138 D56A,N100A M32L, S92A S298A, E333A, K334A, K326A 477 D56A, N100A M32L, S92AS298A, E333A, K334A, K326A, N434W 375 — — K334L 588 — — S298A, E333A,K334A, K326A 511 D56A, N100Y, M32L, S92A S298A, E333A, K334A, S100aRK326A

(iv) Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al, Nature, 362:255-258 (1993); Bruggermann et al, Year inImmuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al, EMBO J.12:725-734 (1993). See also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

(v) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. The antibody fragmentmay also be a “linear antibody”, e.g., as described in U.S. Pat. No.5,641,870 for example. Such linear antibody fragments may bemonospecific or bispecific.

(vi) Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the B cell surface marker. Other suchantibodies may bind the B cell surface marker and further bind a seconddifferent B-cell surface marker. Alternatively, an anti-B cell surfacemarker binding arm may be combined with an arm that binds to atriggering molecule on a leukocyte such as a T-cell receptor molecule(e.g. CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64),FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defensemechanisms to the B cell. Bispecific antibodies may also be used tolocalize cytotoxic agents to the B cell. These antibodies possess a Bcell surface marker-binding arm and an arm that binds the cytotoxicagent (e.g. saporin, anti-interferon-α, vinca alkaloid, ricin A chain,methotrexate or radioactive isotope hapten). Bispecific antibodies canbe prepared as full length antibodies or antibody fragments (e.g.F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO1,10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. In some embodiments,the fusion is with an immunoglobulin heavy chain constant domain,comprising at least part of the hinge, CH2, and CH3 regions. In someembodiments, the first heavy chain constant region (CH1) containing thesite necessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In some embodiments of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal, Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers that are recovered fromrecombinant cell culture. In some embodiments, the interface comprisesat least a part of the C_(H)3 domain of an antibody constant domain. Inthis method, one or more small amino acid side chains from the interfaceof the first antibody molecule are replaced with larger side chains(e.g. tyrosine or tryptophan). Compensatory “cavities” of identical orsimilar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chainswith smaller ones (e.g. alanine or threonine). This provides a mechanismfor increasing the yield of the heterodimer over other unwantedend-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol, 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise a heavychain variable domain (V_(H)) connected to a light chain variable domain(V_(L)) by a linker that is too short to allow pairing between the twodomains on the same chain. Accordingly, the V_(H) and V_(L) domains ofone fragment are forced to pair with the complementary V_(L) and V_(H)domains of another fragment, thereby forming two antigen-binding sites.Another strategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

V. Conjugates and Other Modifications of the Antibody

The antibody used in the methods or included in the articles ofmanufacture herein is optionally conjugated to a cytotoxic agent. Forinstance, the antibody may be conjugated to a drug as described inWO2004/032828.

Chemotherapeutic agents useful in the generation of suchantibody-cytotoxic agent conjugates have been described above.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, a maytansine (U.S. Pat. No. 5,208,020), a trichothene,and CC1065 are also contemplated herein. In one embodiment of theinvention, the antibody is conjugated to one or more maytansinemolecules (e.g. about 1 to about 10 maytansine molecules per antibodymolecule). Maytansine may, for example, be converted to May-SS-Me, whichmay be reduced to May-SH3 and reacted with modified antibody (Chari etal. Cancer Research 52: 127-131 (1992)) to generate amaytansinoid-antibody conjugate.

Alternatively, the antibody is conjugated to one or more calicheamicinmolecules. The calicheamicin family of antibiotics is capable ofproducing double-stranded DNA breaks at sub-picomolar concentrations.Structural analogues of calicheamicin that may be used include, but arenot limited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ₁^(I) (Hinman et al. Cancer Research 53: 3336-3342 (1993) and Lode et al.Cancer Research 58: 2925-2928 (1998)).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates antibody conjugated with acompound with nucleolytic activity (e.g. a ribonuclease or a DNAendonuclease such as a deoxyribonuclease; DNase).

A variety of radioactive isotopes are available for the production ofradioconjugated antibodies. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, dimethyl linker or disulfide-containinglinker (Chari et al. Cancer Research 52: 127-131 (1992)) may be used.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g. by recombinant techniques or peptide synthesis.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g. avidin) thatis conjugated to a cytotoxic agent (e.g. a radionucleotide).

The antibodies of the present invention may also be conjugated with aprodrug-activating enzyme that converts a prodrug (e.g. a peptidylchemotherapeutic agent, see WO81/01145) to an active anti-cancer drug.See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278.

The enzyme component of such conjugates includes any enzyme capable ofacting on a prodrug in such a way so as to covert it into its moreactive, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the antibody bytechniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature, 312: 604-608 (1984)).

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol and polypropylene glycol. In some embodiments, the antibodyfragments, such as Fab′, are linked to one or more PEG molecules.

The antibodies disclosed herein may also be formulated as liposomes.Liposomes containing the antibody are prepared by methods known in theart, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA,82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030(1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 publishedOct. 23, 1997. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of an antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

Amino acid sequence modification(s) of the antibody are contemplated.For example, it may be desirable to improve the binding affinity and/orother biological properties of the antibody. Amino acid sequencevariants of the antibody are prepared by introducing appropriatenucleotide changes into the antibody nucleic acid, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe antibody, such as changing the number or position of glycosylationsites.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody ofan enzyme, or a polypeptide that increases the serum half-life of theantibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by different residue. The sites of greatest interest forsubstitutional mutagenesis of antibody antibodies include thehypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 2 under the heading of“preferred substitutions”. If such substitutions result in a change inbiological activity, then more substantial changes, denominated“exemplary substitutions” in Table 2, may be introduced and the productsscreened.

TABLE 2 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (Q)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), His(H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody. Generally, the resulting variant(s) selected for furtherdevelopment will have improved biological properties relative to theparent antibody from which they are generated. A convenient way forgenerating such substitutional variants is affinity maturation usingphage display. Briefly, several hypervariable region sites (e.g. 6-7sites) are mutated to generate all possible amino substitutions at eachsite. The antibody variants thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedvariants are then screened for their biological activity (e.g. bindingaffinity) as herein disclosed. In order to identify candidatehypervariable region sites for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding. Alternatively, or inadditionally, it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points between the antibodyand antigen. Such contact residues and neighboring residues arecandidates for substitution according to the techniques elaboratedherein. Once such variants are generated, the panel of variants ispatiented to screening as described herein and antibodies with superiorproperties in one or more relevant assays may be selected for furtherdevelopment.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. Such altering includes deletingone or more carbohydrate moieties found in the antibody, and/or addingone or more glycosylation sites that are not present in the antibody.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 A1 (Presta,L.); see also US 2004/0093621 A1 (Kyowa Hakko Kogyo Co., Ltd) concerninga CD20 antibody composition. Antibodies with a bisectingN-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fcregion of the antibody are referenced in WO03/011878, Jean-Mairet et al.and U.S. Pat. No. 6,602,684, Umana et al. Antibodies with at least onegalactose residue in the oligosaccharide attached to an Fc region of theantibody are reported in WO97/30087 (Patel et al.); see also WO98/58964(Raju, S.) and WO99/22764 (Raju, S.) concerning antibodies with alteredcarbohydrate attached to the Fc region thereof.

In some embodiments, the glycosylation variant herein comprises an Fcregion, wherein a carbohydrate structure attached to the Fc region lacksfucose. Such variants have improved ADCC function. Optionally, the Fcregion further comprises one or more amino acid substitutions thereinwhich further improve ADCC, for example, substitutions at positions 298,333, and/or 334 of the Fc region (Eu numbering of residues). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodiesinclude: US Pat. Appl. No. US 2003/0157108 A1, Presta, L; WO 00/61739A1;WO01/29246A1; US2003/0115614A1; US2002/0164328A1; US2004/0093621A1;US2004/0132140A1; US2004/0110704A1; US2004/0110282A1; US2004/0109865A1;WO03/085119A1; WO03/084570A1; WO2005/035778; WO2005/035586 (describingRNA inhibition (RNAi) of fucosylation); Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to modify the antibody of the invention with respectto effector function, e.g. so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of an antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al, J. Exp Med 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered that has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

WO00/42072 (Presta, L.) describes antibodies with improved ADCC functionin the presence of human effector cells, where the antibodies compriseamino acid substitutions in the Fc region thereof. In some embodiments,the antibody with improved ADCC comprises substitutions at positions298, 333, and/or 334 of the Fc region. In some embodiments, the alteredFc region is a human IgG1 Fc region comprising or consisting ofsubstitutions at one, two or three of these positions.

Antibodies with altered Clq binding and/or complement dependentcytotoxicity (CDC) are described in WO99/51642, U.S. Pat. No.6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No. 6,528,624B1 andU.S. Pat. No. 6,538,124 (Idusogie et al.). The antibodies comprise anamino acid substitution at one or more of amino acid positions 270, 322,326, 327, 329, 313, 333 and/or 334 of the Fc region thereof.

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule. Antibodies with substitutions in an Fc region thereofand increased serum half-lives are also described in WO00/42072 (Presta,L.).

Engineered antibodies with three or more (preferably four) functionalantigen binding sites are also contemplated (US Appln No. US2002/0004587A1, Miller et al.).

VI. Pharmaceutical Formulations

Therapeutic formulations of the antibodies used in accordance with thepresent invention are prepared for storage by mixing an antibody havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Exemplary anti-CD20 antibody formulations are described in WO98/56418.This publication describes a liquid multidose formulation comprising 40mg/mL Rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol,0.02% polysorbate 20 at pH 5.0 that has a minimum shelf life of twoyears storage at 2-BBC. Another anti-CD20 formulation of interestcomprises 10 mg/mL Rituximab in 9.0 mg/mL sodium chloride, 7.35 mg/mLsodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Waterfor Injection, pH 6.5.

Lyophilized formulations adapted for subcutaneous administration aredescribed in U.S. Pat. No. 6,267,958 (Andya et al.). Such lyophilizedformulations may be reconstituted with a suitable diluent to a highprotein concentration and the reconstituted formulation may beadministered subcutaneously to the mammal to be treated herein.

Crystalized forms of the antibody or antibody are also contemplated.See, for example, US 2002/0136719A1 (Shenoy et al).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, in someembodiments, those with complementary activities that do not adverselyaffect each other. For example, it may be desirable to further provide acytotoxic agent; chemotherapeutic agent; immunosuppressive agent;cytokine; cytokine antagonist or antibody; growth factor; hormone;integrin; integrin antagonist or antibody (e.g. an LFA-1 antibody suchas efalizumab/RAPTIVA commercially available from Genentech, or an alpha4 integrin antibody such as natalizumab/TYSABRI®) available from BiogenIdec/Elan Pharmaceuticals, Inc.); interferon class drug such asIFN-beta-1a (REBIF® and AVONEX®) or IFN-beta-1b (BETASERON®); anoligopeptide such a glatiramer acetate (COPAXONE®); a cytotoxic agentsuch as mitoxantrone (NOVANTRONE®), methotrexate, cyclophosphamide,chlorambucil, or azathioprine; intravenous immunoglobulin (gammaglobulin); lymphocyte-depleting drug (e.g., mitoxantrone,cyclophosphamide, Campath, anti-CD4, or cladribine);non-lymphocyte-depleting immunosuppressive drug (e.g., mycophenolatemofetil (MMF) or cyclosporine); cholesterol-lowering drug of the“statin” class; estradiol; testosterone; hormone replacement therapy;drug that treats symptoms secondary or related to MS (e.g., spasticity,incontinence, pain, fatigue); a TNF inhibitor; disease-modifyinganti-rheumatic drug (DMARD); non-steroidal anti-inflammatory drug(NSAID); corticosteroid (e.g. methylprednisolone, prednisone,dexamethasone, or glucorticoid); levothyroxine; cyclosporin A;somatastatin analogue; cytokine antagonist; anti-metabolite;immunosuppressive agent; integrin antagonist or antibody (e.g. an LFA-1antibody, such as efalizumab or an alpha 4 integrin antibody such asnatalizumab); or another B-cell surface antagonist/antibody; etc in theformulation. The type and effective amounts of such other agents depend,for example, on the amount of antibody present in the formulation, thetype of multiple sclerosis being treated, and clinical parameters of thepatients. These are generally used in the same dosages and withadministration routes as used hereinbefore or about from 1 to 99% of theheretofore employed dosages.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

In some embodiments, the formulation comprises one or more of the groupconsisting of a histidine buffer, trehalose, sucrose, and polysorbate20. In some embodiments, the histidine buffer is a histidine-acetatebuffer, pH 6.0. Examples of formulations suitable for the administrationof the anti-CD20 antibody are found in Andya et al., US2006/0088523,which is incorporated by reference in its entirety with respect toformulations.

Exemplary anti-CD20 antibody formulations are described in Andya et al.,US2006/0088523 and WO98/56418, which are incorporated by reference inits entirety. In some embodiments, formulation is a liquid multidoseformulation comprising the anti-CD20 antibody at 40 mg/mL, 25 mMacetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 atpH 5.0 that has a minimum shelf life of two years storage at 2-8° C. Insome embodiments, anti-CD20 formulation of interest comprises 10 mg/mLantibody in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citratedihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection, pH6.5. In some embodiments, the anti-CD20 antibody is in an aqueouspharmaceutical formulation comprising 10-30 mM sodium acetate from aboutpH 4.8 to about pH 5.5, preferably at pH5.5, polysorbate as a surfactantin a an amount of about 0.01-0.1% v/v, trehalose at an amount of about2-10% w/v, and benzyl alcohol as a preservative (U.S. Pat. No.6,171,586, which is incorporated by reference in its entirety).Lyophilized formulations adapted for subcutaneous administration aredescribed in WO97/04801, which is incorporated by reference in itsentirety. Such lyophilized formulations may be reconstituted with asuitable diluent to a high protein concentration and the reconstitutedformulation may be administered subcutaneously to the mammal to betreated herein.

In some embodiments, the humanized 2H7 variants formulation is antibodyat 12-14 mg/mL in 10 mM histidine, 6% sucrose, 0.02% polysorbate 20, pH5.8. In a specific embodiment, 2H7 variants and in particular 2H7.v16 isformulated at 20 mg/mL antibody in 10 mM histidine sulfate, 60 mg/mlsucrose., 0.2 mg/ml polysorbate 20, and Sterile Water for Injection, atpH5.8. In a specific embodiment, one IV formulation of humanized 2H7 v16is: 30 mg/ml antibody in 20 mM sodium acetate, 4% trehalose dihydrate,0.02% polysorbate 20 (Tween 20™), pH 5.3. In some embodiments, thehumanized 2H7.v511 variant formulation is 15-30 mg/ml antibody,preferably 20 mg/mL antibody, in 10 mM histidine sulfate, 60 mg/mlsucrose (6%), 0.2 mg/ml polysorbate 20 (0.02%), and Sterile Water forInjection, at pH5.8. In yet another embodiment, the formulation for 2H7variants and in particular 2H7.v511 is 20 mg/ml 2H7, 20 mM sodiumacetate, 4% trehalose dihydrate, 0.02% polysorbate 20, pH 5.5, forintravenous administration. In some embodiments, 2H7.v 114 formulationis antibody at 15-25 mg/ml, preferably 20 mg/ml, in 20 mM SodiumAcetate, 240 mM (8%) trehalose dihydrate, 0.02% Polysorbate 20, pH 5.3.

VII. Articles of Manufacture and Methods of Manufacture

The invention provides articles of manufacture comprising: (a) acontainer comprising ocrelizumab; and (b) a package insert withinstructions for treating multiple sclerosis in a patient, wherein theinstructions denote (i.e., indicate) that an amount of ocrelizumab isadministered to the patient that is effective to provide an initialocrelizumab exposure of between about 0.3 to about 0.6 grams followed bya second ocrelizumab exposure of between about 0.3 to about 0.6 grams,the second exposure not being administered until from about 16 to 60weeks from the initial exposure, and each of the ocrelizumab exposuresis provided to the patient as one or two doses of ocrelizumab. In someembodiments, the initial ocrelizumab exposure is about 0.6 grams. Insome embodiments, the second ocrelizumab exposure is about 0.6 grams. Insome embodiments, the second exposure is administered from about 24weeks from the initial exposure. In some embodiments, one or more of theocrelizumab exposures are provided to the patient as one dose ofocrelizumab. In some embodiments, one or more of the ocrelizumabexposures are provided to the patient as two doses of ocrelizumab. Insome embodiments, the two doses of ocrelizumab comprise about 0.3 gramsof ocrelizumab.

The invention further provides articles of manufacture containingmaterials useful for the treatment of progressive multiple sclerosisdescribed herein. In some embodiments, the article of manufacturecomprising, packaged together, a pharmaceutical composition comprisingan anti-CD20 antibody and a pharmaceutically acceptable carrier and alabel denoting that the anti-CD20 antibody or pharmaceutical compositionis indicated for treating patients with multiple sclerosis having one ormore characteristics selected from the group consisting of (a) an ageless than about 55 years, (b) one or more gadolinium staining lesions,(c) at least about a one point increase in Expanded Disability StatusScale (EDSS) over two years prior to starting the treatment, and (d) aMultiple Sclerosis Severity Score (MSSS) greater than about 5 points.

In some embodiments, the article of manufacture comprising, packagedtogether, a pharmaceutical composition comprising an anti-CD20 antibodyand a pharmaceutically acceptable carrier and a label denoting thatadministration of the anti-CD20 antibody or pharmaceutical compositionis based upon the patient having one or more characteristics selectedfrom the group consisting of (a) an age less than about 55 years, (b)one or more gadolinium staining lesions, (c) at least about a one pointincrease in Expanded Disability Status Scale (EDSS) over two years priorto starting treatment, and (d) a Multiple Sclerosis Severity Score(MSSS) greater than about 5 points.

In some embodiments, the article of manufacture comprising, packagedtogether, a pharmaceutical composition comprising an anti-CD20 antibodyand a pharmaceutically acceptable carrier and a label denoting that thepharmaceutical composition is administered to a selected patient,wherein the selected patient has one or more characteristics selectedfrom the group consisting of (a) an age less than about 55 years, (b)one or more gadolinium staining lesions, (c) at least about a one pointincrease in Expanded Disability Status Scale (EDSS) over two years priorto starting treatment, and (d) a Multiple Sclerosis Severity Score(MSSS) greater than about 5 points.

In some embodiments of any of the articles of manufacture describedherein, the patients have one or more characteristics selected from thegroup consisting of (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, and (c) at least about a one point increasein Expanded Disability Status Scale (EDSS) over two years prior tostarting treatment.

In some embodiments of any of the articles of manufacture describedherein, the patient has more than one characteristics selected from thegroup consisting of (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, (c) at least about a one point increase inExpanded Disability Status Scale (EDSS) over two years prior to startingtreatment, and (d) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points. In some embodiments of any of the articles ofmanufacture described herein, the patient has two characteristicsselected from the group consisting of (a) an age less than about 55years, (b) one or more gadolinium staining lesions, and (c) at leastabout a one point increase in EDSS over two years prior to startingtreatment. In some embodiments, the patient has three characteristicsselected from the group consisting of (a) an age less than about 55years, (b) one or more gadolinium staining lesions, (c) at least about aone point increase in Expanded Disability Status Scale (EDSS) over twoyears prior to starting treatment, and (d) a Multiple Sclerosis SeverityScore (MSSS) greater than about 5 points. In some embodiments, thepatient has (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, (c) at least about a one point increase inExpanded Disability Status Scale (EDSS) over two years prior to startingtreatment, and (d) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points.

In some embodiments of any of the articles of manufacture, theprogressive multiple sclerosis is primary progressive multiplesclerosis. In some embodiments, the progressive multiple sclerosis issecondary progressive multiple sclerosis. In some embodiments, theprogressive multiple sclerosis is progressive relapsing multiplesclerosis. In some embodiments, the patient is not diagnosed withrelapsing remitting multiple sclerosis when starting treatment.

In some embodiments of any of the articles of manufacture, the patientfurther has evidence of inflammation in a sample. In some embodiments,the sample is a cerebrospinal fluid sample. In some embodiments, theevidence of inflammation is indicated by an elevated IgG index. In someembodiments, the evidence of inflammation is indicated by IgGoligoclonal bands detected by isoelectric focusing.

In some embodiments of any of the articles of manufacture, the increasein EDSS over two years prior to starting treatment is not attributableto relapse. In some embodiments, the patient has had an EDSS of greaterthan about 5.0 for less than about 15 years. In some embodiments, thepatient has had an EDSS less than or equal to about 5.0 for less thanabout 10 years. In some embodiments, the EDSS when starting treatment isbetween about 3.0 and about 6.5. In some embodiments, the increase inEDSS is at least about a 1.5 point increase in EDSS over two years priorto starting treatment. In some embodiments, the 1.5 point increase inEDSS over two years prior to starting treatment is not attributable torelapse. In some embodiments, the patient further had two or morerelapses within two years prior to starting treatment.

In some embodiments of any of the articles of manufacture describedherein, the patient and/or patient population is characterized by havingan MSSS of greater than about any of 6, 7, 8, or 9. In some embodimentsof any of the articles of manufacture, the patient and/or patientpopulation is characterized by having an MSSS of greater than about 9.

In some embodiments of any of the articles of manufacture, the age ofthe patient is less than about 51.

In some embodiments of any of the articles of manufacture, the treatmentreduces the time to confirmed disease progression. In some embodiments,the confirmed disease progression is an increase in EDSS that issustained for twelve weeks. In some embodiments, the confirmed diseaseprogression is an increase in EDSS that is sustained for twenty-fourweeks.

In some embodiments of any of the articles of manufacture, the anti-CD20antibody comprises: a) a heavy chain variable region comprising SEQ IDNO:10, SEQ ID NO:11, and SEQ ID NO:12, and b) a light chain variableregion comprising SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In someembodiments, the anti-CD20 antibody is ocrelizumab. In some embodiments,the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20antibody is ofatumumab. In some embodiments, the anti-CD20 antibody isTRU-015 or SBI-087. In some embodiments, the anti-CD20 antibody isGA101. In some embodiments, the anti-CD20 antibody is hA20.

The article of manufacture comprises a container and a label or packageinsert on or associated with the container. Suitable containers include,for example, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds or contains a composition that is effective for treating themultiple sclerosis and may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the composition is the antibody. In some embodiments, the containercomprises between about 0.3 to about 4.0 grams of the anti-CD20antibody. In some embodiments, the container comprises between about 0.3to about 1.5 grams of the anti-CD20 antibody.

The label or package insert indicates that the composition is used fortreating multiple sclerosis in a patient suffering therefrom withspecific guidance regarding dosing amounts and intervals of antibody andany other drug being provided. The article of manufacture may furthercomprise a second container comprising a pharmaceutically acceptablediluent buffer, such as bacteriostatic water for injection (BWFI),phosphate-buffered saline, Ringer's solution and dextrose solution. Thearticle of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

Optionally, the article of manufacture herein further comprises acontainer comprising an agent other than the antibody for treatment andfurther comprising instructions on treating the patient with such agent,such agent preferably being a chemotherapeutic agent orimmunosuppressive agent, interferon class drug such as IFN-beta-1a(REBIF® and AVONEX®) or IFN-beta-1b (BETASERON®); an oligopeptide such aglatiramer acetate (COPAXONE®); a cytotoxic agent such as mitoxantrone(NOVANTRONE®), methotrexate, cyclophosphamide, chlorambucil, orazathioprine; intravenous immunoglobulin (gamma globulin);lymphocyte-depleting drug (e.g., mitoxantrone, cyclophosphamide,Campath, anti-CD4, or cladribine); non-lymphocyte-depletingimmunosuppressive drug (e.g., mycophenolate mofetil (MMF) orcyclosporine); cholesterol-lowering drug of the “statin” class;estradiol; hormone replacement therapy; drug that treats symptomssecondary or related to MS (e.g., spasticity, incontinence, pain,fatigue); a TNF inhibitor; disease-modifying anti-rheumatic drug(DMARD); non-steroidal anti-inflammatory drug (NSAID); corticosteroid(e.g. methylprednisolone, prednisone, dexamethasone, or glucorticoid);levothyroxine; cyclosporin A; somatastatin analogue; cytokine orcytokine receptor antagonist; anti-metabolite; immunosuppressive agent;integrin antagonist or antibody (e.g. an LFA-1 antibody, such asefalizumab or an alpha 4 integrin antibody such as natalizumab); andanother B-cell surface marker antibody; etc.

In some embodiments, the label may further denote any of the embodimentsdescribed herein. For example, the label may denote that the patient has(a) an age less than about 55 years and (b) one or more gadoliniumstaining lesions.

In another embodiment of the invention, a method for manufacturingcontaining materials useful for the treatment of progressive multiplesclerosis described herein is provided. In some embodiments, the methodfor manufacturing an anti-CD20 antibody or a pharmaceutical compositionthereof comprising combining in a package the anti-CD20 antibody orpharmaceutical composition and a label denoting that the anti-CD20antibody or pharmaceutical composition is indicated for treatingpatients with progressive multiple sclerosis, wherein the patients haveone or more characteristics selected from the group consisting of (a) anage less than about 55 years, (b) one or more gadolinium staininglesions, (c) at least about a one point increase in Expanded DisabilityStatus Scale (EDSS) over two years prior to starting treatment, and (d)a Multiple Sclerosis Severity Score (MSSS) greater than about 5 points.

In some embodiments of any of the methods of manufacturing describedherein, the patients have one or more characteristics selected from thegroup consisting of (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, and (c) at least about a one point increasein Expanded Disability Status Scale (EDSS) over two years prior tostarting treatment.

VIII. Methods of Advertising and Marketing

The present invention also provides methods for advertising an anti-CD20antibody or a pharmaceutically acceptable composition thereof comprisingpromoting, to a target audience, the use of the anti-CD20 antibody orpharmaceutical composition thereof for treating a patient or patientpopulation with progressive multiple sclerosis, wherein the patient orpatient population has one or more characteristics selected from thegroup consisting of (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, (c) at least about a one point increase inExpanded Disability Status Scale (EDSS) over two years prior to startingtreatment, and (d) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points.

Provided herein are also methods for marketing an anti-CD20 antibody ora pharmaceutically acceptable composition thereof for use in aprogressive multiple sclerosis patient subpopulation, the methodcomprising informing a target audience about the use of the anti-CD20antibody for treating the patient subpopulation characterized by thepatients of such subpopulation having one or more characteristicsselected from the group consisting of (a) an age less than about 55years, (b) one or more gadolinium staining lesions, (c) at least about aone point increase in Expanded Disability Status Scale (EDSS) over twoyears prior to starting treatment, and (d) a Multiple Sclerosis SeverityScore (MSSS) greater than about 5 points.

In addition, the invention provides methods of specifying an anti-CD20antibody for use in a progressive multiple sclerosis patientsubpopulation, the method comprising providing instruction to administerthe anti-CD20 antibody or a pharmaceutically acceptable compositionthereof to the patient subpopulation characterized by the subpopulationhas one or more characteristics selected from the group consisting of(a) an age less than about 55 years, (b) one or more gadolinium staininglesions, (c) at least about a one point increase in Expanded DisabilityStatus Scale (EDSS) over two years prior to starting treatment, and (d)a Multiple Sclerosis Severity Score (MSSS) greater than about 5 points.

The invention further provides methods of providing a treatment optionfor patients with progressive multiple sclerosis comprising packaging ananti-CD20 antibody in a vial with a package insert containinginstructions to treat patients with progressive multiple sclerosis,wherein the patients have one or more characteristics selected from thegroup consisting of (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, (c) at least about a one point increase inExpanded Disability Status Scale (EDSS) over two years prior to startingtreatment, and (d) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points.

In some embodiments of any of the methods, the patients have one or morecharacteristics selected from the group consisting of (a) an age lessthan about 55 years, (b) one or more gadolinium staining lesions, and(c) at least about a one point increase in Expanded Disability StatusScale (EDSS) over two years prior to starting treatment.

In some embodiments of any of the methods, the patient has twocharacteristics selected from the group consisting of (a) an age lessthan about 55 years, (b) one or more gadolinium staining lesions, (c) atleast about a one point increase in Expanded Disability Status Scale(EDSS) over two years prior to starting treatment, and (d) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points. In someembodiments, the patient has three characteristics selected from thegroup consisting of (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, (c) at least about a one point increase inExpanded Disability Status Scale (EDSS) over two years prior to startingtreatment, and (d) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points. In some embodiments, the patient has (a) an ageless than about 55 years, (b) one or more gadolinium staining lesions,(c) at least about a one point increase in Expanded Disability StatusScale (EDSS) over two years prior to starting treatment, and (d) aMultiple Sclerosis Severity Score (MSSS) greater than about 5 points.

In some embodiments of any of the methods, the progressive multiplesclerosis is primary progressive multiple sclerosis. In someembodiments, the progressive multiple sclerosis is secondary progressivemultiple sclerosis. In some embodiments, the progressive multiplesclerosis is progressive relapsing multiple sclerosis. In someembodiments, the patient is not diagnosed with relapsing remittingmultiple sclerosis when starting treatment.

In some embodiments of any of the methods, the patient further hasevidence of inflammation in a sample. In some embodiments, the sample isa cerebrospinal fluid sample. In some embodiments, the evidence ofinflammation is indicated by an elevated IgG index. In some embodiments,the evidence of inflammation is indicated by IgG oligoclonal bandsdetected by isoelectric focusing.

In some embodiments of any of the methods, the increase in EDSS over twoyears prior to starting treatment is not attributable to relapse. Insome embodiments, the patient has had an EDSS of greater than about 5.0for less than about 15 years. In some embodiments, the patient has hadan EDSS less than or equal to about 5.0 for less than about 10 years. Insome embodiments, the EDSS when starting treatment is between about 3.0and about 6.5. In some embodiments, the increase in EDSS is at leastabout a 1.5 point increase in EDSS over two years prior to startingtreatment. In some embodiments, the 1.5 point increase in EDSS over twoyears prior to starting treatment is not attributable to relapse. Insome embodiments, the patient further had two or more relapses withintwo years prior to starting treatment.

In some embodiments of any of the methods, the age of the patient isless than about 51.

In some embodiments of any of the methods, the treatment reduces thetime to confirmed disease progression. In some embodiments, theconfirmed disease progression is an increase in EDSS that is sustainedfor twelve weeks. In some embodiments, the confirmed disease progressionis an increase in EDSS that is sustained for twenty-four weeks.

In some embodiments of any of the methods, the anti-CD20 antibody isocrelizumab. In some embodiments of any of the methods, the anti-CD20antibody is rituximab. In some embodiments of any of the methods, theanti-CD20 antibody is ofatumumab. In some embodiments of any of themethods, the anti-CD20 antibody is TRU-015 or SBI-087. In someembodiments of any of the methods, the anti-CD20 antibody is GA101. Insome embodiments of any of the methods, the anti-CD20 antibody is hA20.

The methods described herein may encompass any combination of theembodiments described herein. For example, the methods include methods,wherein the patient (a) an age less than about 55 years and (b) one ormore gadolinium staining lesions.

IX. Systems and Methods for Predicting Responsiveness to MultipleSclerosis Treatment

The invention also provides systems and methods for analyzing whether asubject and/or patient with progressive multiple sclerosis will respondto a treatment with a drug used to treat multiple sclerosis. Theinvention provides systems for analyzing responsiveness of a patientwith progressive multiple sclerosis to treatment with a drug used totreat multiple sclerosis comprising: (a) assessing one or morecharacteristics selected from the group consisting of (i) an age lessthan about 55 years, (ii) one or more gadolinium staining lesions, (iii)at least about a one point increase in Expanded Disability Status Scale(EDSS) over two years prior to starting treatment, and (iv) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points; (b)hardware to perform the assessment of (a); and (c) computational meansto perform an algorithm to determine if the patient is susceptible orresponsive to said treatment.

The invention further provides methods for predicting whether a subjectwith progressive multiple sclerosis will respond to a treatment with adrug used to treat multiple sclerosis, the methods comprising assessingone or more characteristics selected from the group consisting of (a) anage less than about 55 years, (b) one or more gadolinium staininglesions, (c) at least about a one point increase in Expanded DisabilityStatus Scale (EDSS) over two years prior to starting treatment, and (d)a Multiple Sclerosis Severity Score (MSSS) greater than about 5 points,whereby the age, the gadolinium staining lesions, the increase in EDDSover two years prior to starting the treatment, MSSS, or a combinationthereof indicates that the subject will respond to the treatment.

In some embodiments of any of the systems and/or methods, the patienthas more than one characteristics selected from the group consisting of(a) an age less than about 55 years, (b) one or more gadolinium staininglesions, and (c) at least about a one point increase in ExpandedDisability Status Scale (EDSS) over two years prior to startingtreatment.

In some embodiments of any of the systems and/or methods describedherein, the patient has more than one characteristics selected from thegroup consisting of (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, (c) at least about a one point increase inExpanded Disability Status Scale (EDSS) over two years prior to startingtreatment, and (d) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points. In some embodiments of any of the systems and/ormethods, the patient has two characteristics selected from the groupconsisting of (a) an age less than about 55 years, (b) one or moregadolinium staining lesions, (c) at least about a one point increase inExpanded Disability Status Scale (EDSS) over two years prior to startingtreatment, and (d) a Multiple Sclerosis Severity Score (MSSS) greaterthan about 5 points. In some embodiments, the patient has threecharacteristics selected from the group consisting of (a) an age lessthan about 55 years, (b) one or more gadolinium staining lesions, (c) atleast about a one point increase in Expanded Disability Status Scale(EDSS) over two years prior to starting treatment, and (d) a MultipleSclerosis Severity Score (MSSS) greater than about 5 points. In someembodiments, the patient has (a) an age less than about 55 years, (b)one or more gadolinium staining lesions, (c) at least about a one pointincrease in Expanded Disability Status Scale (EDSS) over two years priorto starting treatment, and (d) a Multiple Sclerosis Severity Score(MSSS) greater than about 5 points.

In some embodiments of any of the systems and/or methods, theprogressive multiple sclerosis is primary progressive multiplesclerosis. In some embodiments, the progressive multiple sclerosis issecondary progressive multiple sclerosis. In some embodiments, theprogressive multiple sclerosis is progressive relapsing multiplesclerosis. In some embodiments, the patient is not diagnosed withrelapsing remitting multiple sclerosis when starting treatment.

In some embodiments of any of the systems or methods, the patientfurther has evidence of inflammation in a sample. In some embodiments,the sample is a cerebrospinal fluid sample. In some embodiments, theevidence of inflammation is indicated by an elevated IgG index. In someembodiments, the evidence of inflammation is indicated by IgGoligoclonal bands detected by isoelectric focusing.

In some embodiments of any of the systems and/or methods, the increasein EDSS over two years prior to starting treatment is not attributableto relapse. In some embodiments, the patient has had an EDSS of greaterthan about 5.0 for less than about 15 years. In some embodiments, thepatient has had an EDSS less than or equal to about 5.0 for less thanabout 10 years. In some embodiments, the EDSS when starting treatment isbetween about 3.0 and about 6.5. In some embodiments, the increase inEDSS is at least about a 1.5 point increase in EDSS over two years priorto starting treatment. In some embodiments, the 1.5 point increase inEDSS over two years prior to starting treatment is not attributable torelapse. In some embodiments, the patient further had two or morerelapses within two years prior to starting treatment.

In some embodiments of any of the systems and/or methods, the age of thepatient is less than about 51.

In some embodiments of any of the systems and/or methods, the systems ormethods further comprises advising the patient.

In some embodiments of any of the systems and/or methods, the treatmentreduces the time to confirmed disease progression. In some embodiments,the confirmed disease progression is an increase in EDSS that issustained for twelve weeks. In some embodiments, the confirmed diseaseprogression is an increase in EDSS that is sustained for twenty-fourweeks.

In some embodiments of any of the systems and/or methods, the drug is aInterferon beta-1b (e.g., Betaseron®), Interferon beta-la (e.g., Avonex®or Rebif®), Glatiramer (e.g., Copaxone®), Mitoxantrone (e.g.,Novantrone), corticosteroids (e.g., ethylprednisolone, prednisone,dexamethasone), 3-4 diaminopyridine, ABT-874, Alemtuzumab, Albuterol(Proventil®), ATL1102, Atorvastatin (Lipitor®), Azathioprine, BG00012(dimethyl fumarate), BHT-3009, Botulinum toxin A (Botox®), C-105,cannador, dronabinol, tetrahydrocannabinol, cannabidiol, CDP323,Cladribine, CNTO 1275, Cyclophosphamide, Daclizumab,Dextromethorphan/quinidine (AVP-923, Zenvia™), Donepezil (Aricept®),Doxycycline, Estradiol, Estriol, Estroprogestins, Fampridine-SR(4-aminopyridine, sustained release), Fingolimod (FTY720), Interferontau, Lamotrigine (Lamictal®), Laquinimod, Lidocaine+prilocaine (EMLA),MBP8298 (synthetic myelin basic protein peptide), Memantine (Namenda®),Methylprednisolone, MN-166, Modafinil (Provigil®), Mycophenolate mofetil(Cellcept®), naltrexone, Natalizumab (Tysabri®), Paroxetine (Paxil®),PI-2301 (copolymer), Pioglitazone (Actos®), Pixantrone (BBR 2778),Pravastatin (Pravachol®), Pregabalin (Lyrica®), Progesterone, RG2077,Riluzole (Rilutek®), Rolipram (phosphodiesterase-4 inhibitor), RTL1000,SB-683699, Simvastatin (Zocor®), T cell receptor peptide vaccine(NeuroVax™), Teriflunomide, Testosterone gel (Androgel®), orTrimethoprim.

In some embodiments of any of the systems and/or methods, the drug usedto treat multiple sclerosis is an anti-CD20 antibody. In someembodiments, the anti-CD20 antibody comprises: a) a heavy chain variableregion comprising SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, and b) alight chain variable region comprising SEQ ID NO:4, SEQ ID NO:5, and SEQID NO:6. In some embodiments, the anti-CD20 antibody is ocrelizumab. Insome embodiments, the anti-CD20 antibody is rituximab. In someembodiments, the anti-CD20 antibody is ofatumumab. In some embodiments,the anti-CD20 antibody is TRU-015 or SBI-087. In some embodiments, theanti-CD20 antibody is GA101. In some embodiments, the anti-CD20 antibodyis hA20.

The systems and/or methods described herein may encompass anycombination of the embodiments described herein. For example, themethods include methods, wherein the patient (a) an age less than about55 years and (b) one or more gadolinium staining lesions.

Further details of the invention are illustrated by the followingnon-limiting Examples. The disclosures of all citations in thespecification are expressly incorporated herein by reference.

EXAMPLES

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. The foregoing examples and detailed description areoffered by way of illustration and not by way of limitation.

Example 1: A Phase II Study of Ocrelizumab in Relapsing-RemittingMultiple Sclerosis (RRMS)

A phase II, multicenter, randomized, parallel-group, partially blinded,placebo and Avonex controlled dose finding study to evaluate theefficacy as measured by brain Magnetic Resonance Imaging (MRI) lesions,and safety of two dose regimens of ocrelizumab in patients withRelapsing-Remitting Multiple Sclerosis (RRMS) is performed.

The two ocrelizumab dose regimens under investigation are as follows: 1)ocrelizumab 1000 mg dose regimen: consisting of a dual infusion of 1000mg for the first treatment cycle followed by single infusions of 1000 mgfor the subsequent treatment cycles and 2) ocrelizumab 600 mg doseregimen: consisting of a dual infusion of 300 mg for the first treatmentcycle followed by single infusions of 600 mg for the subsequenttreatment cycles.

Eligible patients are randomized (1:1:1:1) into one of four treatmentgroups A, B, C or D as described in FIG. 7. The overview of the studydesign is illustrated in FIG. 7.

Group A (ocrelizumab 1000 mg): Two intravenous (i.v.) infusions ofocrelizumab each of 1000 mg separated by 14 days for the 1st treatmentcycle. Patients then receive the maintenance dose regimen, i.e., asingle infusion of 1000 mg for every subsequent 24-week treatment cycle.Subsequently, in order to maintain the study double-blind during the 2ndtreatment cycle, patients receive two infusions separated by 14 days,the first infusion is ocrelizumab 1000 mg and the second infusionplacebo. In the 3rd and 4th treatment cycles, patients are treated witha single 1000 mg infusion, without a second placebo infusion, in adouble-blind manner until a preferred dose is chosen on the basis of theprimary analysis.

Group B (ocrelizumab 600 mg): Two i.v. infusions of ocrelizumab each of300 mg separated by 14 days for the 1st treatment cycle. Patients thenreceive the maintenance dose regimen, i.e., a single infusion of 600 mgfor every subsequent 24-week treatment cycle. Subsequently, in order tomaintain the study double-blind during the 2nd treatment cycle, patientsreceive two infusions separated by 14 days, the first infusion isocrelizumab 600 mg and the second infusion placebo. In the 3rd and 4thtreatment cycles, patients are treated with a single 600 mg infusion,without a second placebo infusion in a double-blind manner until apreferred dose is chosen on the basis of the primary analysis.

Group C (placebo): Two i.v. infusions of placebo separated by 14 daysfor the 1st treatment cycle. Thereafter, patients are placed on the 600mg dose regimen of ocrelizumab starting with two double-blind i.v.infusions of ocrelizumab 300 mg separated by 14 days at the start of the2nd treatment cycle. Patients then receive the maintenance dose regimen,i.e., a single infusion of 600 mg administered in a double-blind mannerfor the 3rd and 4th treatment cycles, until a preferred dose is chosenon the basis of the primary analysis.

Group D (Avonex): Avonex 30 μg intra-muscular (i.m.) weekly for the 1sttreatment cycle. Thereafter, patients are offered, on a voluntary andopen label basis, the 600 mg dose regimen of ocrelizumab starting withtwo i.v. infusions of ocrelizumab 300 mg separated by 14 days at thestart of the 2nd treatment cycle. Patients in the 3rd and 4th treatmentcycles are treated with a single 600 mg infusion until a preferred doseis chosen on the basis of the primary analysis.

For all groups, after investigators and ethics committees are informedof the preferred dose, patients receive the preferred dose (600 mg or1000 mg) as a single infusion at their next successive treatmentcycle(s).

The first administration of study medication whether an i.v. infusion(ocrelizumab or placebo) or the first i.m. injection of Avonex willdefine the start of the Treatment Period (Day 1). All patients alsoreceive an i.v. infusion of methylprednisolone 100 mg on study day 1 andwith each subsequent ocrelizumab or placebo infusion or, for patientsreceiving Avonex (Group D) according to the time points required for theocrelizumab infusions.

There are four treatment cycles, i.e. cycle 1=Baseline to Week 24; cycle2=Week 24 to Week 48; cycle 3=Week 48 to Week 72; cycle 4=Week 72 toWeek 96. After the 1st cycle infusion visits (Visits 2 and 3, Day 1 andWeek 2, respectively), visits occur at Week 4 and every 4 weeksthereafter for the first 24 weeks. After the 2nd cycle infusion visits(Visits 9 and 10, Week 24 and 26, respectively), visits occur at Week 36and every 12 weeks thereafter through the end of the Treatment andFollow-up Periods. All effort should be made to schedule the visitswithin the provided windows. Additional unscheduled visits for theassessment of potential relapses, new neurological symptoms or safetyevents may occur at any time.

Study Population and Selection Criteria

Men and women from 18 to 55 years of age inclusive, who are diagnosedwith relapsing-remitting multiple sclerosis (RRMS) in accordance withthe revised McDonald criteria (2005) and who meet theinclusion/exclusion criteria provided below are eligible for enrollmentinto the study.

Inclusion Criteria:

Patients must meet the following criteria to be eligible for studyentry:

-   -   1. RRMS in accordance with the revised McDonald criteria (2005);    -   2. Ages 18-55 years inclusive;    -   3. At least two documented relapses within the last 3 years        prior to screening, at least one of which occurred within the        last year prior to screening;    -   4. Expanded Disability Status Scale (EDSS) at baseline from 1.0        to 6.0 points;    -   5. Evidence of Multiple Sclerosis (MS) disease burden as defined        below:        -   a. At least six T2 lesions on an MRI scan done in the year            prior to screening, based on local reading. Should an MRI            scan be unavailable within the last year or showing less            than six T2 lesions, a screening MRI scan with at least six            T2 lesions is required for the patient to be eligible, OR        -   b. Patient had 2 documented relapses within the year prior            to screening.

Exclusion Criteria

Patients who meet the following criteria will be excluded from studyentry:

1. Secondary or primary progressive multiple sclerosis at screening(Visit 1);

2. Disease duration of more than 15 years in patients with an EDSS≤2.0.

Efficacy Analysis

The primary objective in this study is to investigate the effect ofocrelizumab given as two dose regimens of 600 or 1000 mg intravenously(see FIG. 7) on the total number of gadolinium-enhancing T1 lesionsobserved on MRI scans of the brain at weeks 12, 16, 20 and 24 ascompared with placebo.

The secondary objectives of this study are to evaluate the efficacy andsafety of ocrelizumab compared with placebo, as reflected by thefollowing: the annualized protocol defined relapse rate by Week 24;proportion of patients who remain relapse-free by Week 24 (protocoldefined relapses); the total number of gadolinium-enhancing T1 lesionsobserved on MRI scans of the brain at Weeks 4, 8, 12, 16, 20 and 24; thetotal number of new and/or persisting gadolinium-enhancing T1 lesions onMRI scans of the brain at Weeks 4, 8, 12, 16, 20 and 24; change in totalvolume of T2 lesions on MRI scans of the brain from baseline to Week 24,to evaluate the safety and tolerability of two dose regimens ofocrelizumab in patients with RRMS as compared with placebo and Avonex atWeek 24 and the overall safety of ocrelizumab administered for up to 96weeks, and to investigate the pharmacokinetics and other pharmacodynamicstudy endpoints of ocrelizumab.

In this Example, a relapse is defined as the occurrence of new orworsening neurological symptoms attributable to MS and immediatelypreceded by a relatively stable or improving neurological state of least30 days. Symptoms must persist for >24 hours and should not beattributable to confounding clinical factors (e.g., fever, infection,injury, adverse reactions to concomitant medications). The new orworsening neurological symptoms must be accompanied by objectiveneurological worsening consistent with an increase of at least half astep on the EDSS, or 2 points on one of the appropriate FunctionalSystem Scores (FSS), or 1 point on two or more of the appropriate FSS.The change must affect the selected FSS (i.e., pyramidal, gait,cerebellar, brainstem, sensory, or visual). Sensory changes, episodicspasms, fatigue, mood change or bladder or bowel urgency or incontinencedo not suffice to establish a relapse. The examining investigatorconfirms those relapses that adhere to the above criteria.

The exploratory objectives in this study will include, but may not belimited to: change in brain volume on MRI scans of the brain from thebaseline scan to Week 12; change in brain volume on MRI scans of thebrain from Week 12 to Week 96 in a subgroup of patients receivingocrelizumab; the total number of new and/or enlarging T2 lesionsobserved on MRI scans of the brain at Weeks 4, 8, 12, 16, 20 and 24; theproportion of patients who remain free of new gadolinium-enhancing T1lesions by Week 24; time to first new gadolinium-enhancing T1 lesionsdeveloping over 24 weeks; to evaluate the treatment withdrawal effect bymeans of the total number of gadolinium-enhancing T1 lesions 48 weeksafter receiving up to 4 treatment cycles of ocrelizumab in a subgroup ofpatients; proportion of patients who remain free from relapses (clinicaland protocol-defined relapses) during each treatment cycle and at Weeks48 and 96; proportion of patients requiring systemic methylprednisolonetreatment for an MS relapse during each treatment cycle and at Weeks 48and 96; annualized clinical and protocol-defined relapse rate duringeach treatment cycle and at Weeks 48 and 96; time to firstprotocol-defined relapse by Week 24; time to first protocol-definedrelapse by Week 96; time to onset of sustained disability progression asdefined by the sustained worsening in EDSS of 1.0 point or more for 12weeks through Week 96; time to onset of sustained disability progressionas defined by the sustained worsening in EDSS of 1.0 point or more for24 weeks through Week 96; to explore the effects of ocrelizumab on theprimary and secondary study endpoints vs. Avonex; to explore thecorrelation of polymorphic variants in genes associated with RRMSsusceptibility and ocrelizumab activity and therapeutic response toocrelizumab in RRMS patients; to explore the relationship betweencirculating biomarkers associated with RRMS susceptibility andocrelizumab activity and therapeutic response to ocrelizumab treatmentin RRMS patients; change in the Modified Fatigue Impact Scale (MFIS)from baseline to Weeks 24 and 48; change in the Fatigue Scale for Motorand Cognitive Functions (FSMC) from baseline to Weeks 24 and 48; changein the proportion of patients who moved from “severe” to “moderate” andfrom “moderate” to “mild” fatigue on the FSMC, comparing baseline toWeeks 24 and 48; change in the Center for EpidemiologicalStudies-Depression Scale (CES-D) from baseline to Weeks 24 and 48; andchange in the proportion of patients who moved from a state of greaterdepressive symptomatology to a state of less depressive symptomatologyon the CES-D, comparing baseline to Weeks 24 and 48.

Brain MRI

MRI is a useful tool for monitoring central nervous system (CNS) lesionsin MS. Brain MRI scans are only obtained at screening in some patients(see Secondary Endpoints) and in all patients at baseline and atfour-week intervals between baseline and week 24. In addition, in asubgroup of patients (Groups A and B), a brain MRI scan is performed atweeks 96 (Visit 16) and 48 weeks later, i.e., week 144.

The MRI includes the acquisition of the following scans at each timepoint: T2-weighted MRI scan, T1-weighted MRI scan (withoutgadolinium-enhancement), and T1-weighted MRI scan (withgadolinium-enhancement).

Assessment of Disability

Disability progression as measured by EDSS is assessed in all patientsby the independent examining investigator at screening and every 12weeks throughout the study until the Observation Period at which timedisability progression is assessed after 24 weeks.

Disability progression is defined as an increase of ≥1.0 point from thebaseline EDSS score that is not attributable to another etiology (e.g.fever, concurrent illness, or concomitant medication) when the baselinescore is 5.0 or less, and ≥0.5 when the baseline score is 5.5 or more.Disease progression is considered sustained when the increase in theEDSS is confirmed at a regularly scheduled visit at least 12 weeks afterthe initial documentation of the progression. An alternative definitionof sustained disability progression requires that the increase in EDSSbe confirmed at least 24 weeks after the initial documentation of theprogression.

The EDSS is based on a standard neurological examination; the sevencategories of the EDSS representing functional systems (pyramidal,cerebellar, brainstem, sensory, bowel and bladder, visual, and cerebraland/or mental, plus “other”) are rated and scored (collectively,functional system scores or FSS). Each score of the FSS is an ordinalclinical rating scale ranging from 0 to 5 or 6. These ratings are thenused in conjunction with observations and information concerningambulation and use of assistive devices to determine the EDSS score. TheEDSS is a disability scale that ranges in 0.5-point steps from 0(normal) to 10 (death).

Example 2: A Phase II/III Study of Rituximab in Primary ProgressiveMultiple Sclerosis (PPMS)

A randomized, double-blind, parallel group, placebo controlled,multicenter Phase II/III study (U2786g) to evaluate the safety andefficacy of rituximab in adults with Primary Progressive MultipleSclerosis (PPMS) as defined by McDonald et al (Ann Neurol50:121-7(2001)) was performed.

Subjects were randomized in a 2:1 ratio to receive either rituximab orplacebo. Rituximab, commercially available from Genentech, wasformulated for i.v. administration as a sterile product in 9.0 mg/mlsodium chloride, 0.7 mg/ml polysorbate 80, 7.35 mg/ml sodium citratedehydrate, and sterile water for injection (pH 6.5). Each course ofstudy drug consisted of two i.v. infusions (separated by 14 days) of1000 mg rituximab or placebo. Subjects received the first course oftreatment at Days 1 and 15 and received additional courses at Weeks 24,48, and 72. Subjects received acetaminophen (1 g) and diphenhydramineHCl (50 mg), or equivalent, by mouth 30-60 min prior to start of eachinfusion. Glucocorticoids were not administered prior to infusion. In 96weeks of trial duration, subjects were seen at regularly scheduledvisits for physical examinations, neurologic and MRI assessments, tocollect adverse events and vital signs, and to complete routinehematology, serum chemistries, and urinalysis lab tests.

Baseline demographics of Intent-To-Treat (ITT) subjects are presented inTable 3.

TABLE 3 Demographic and Baseline Characteristics: Intent-to-TreatSubjects. Placebo Rituximab All Subjects Characteristic (n = 147) (n =292) (n = 439) Age (yr) n 147 292 439 Mean (SD) 49.6 (8.69) 50.1 (9.02)49.9 (8.90) Median 51.0 51.0 51.0 Minimum to 20-66 18-66 18-66 maximum18-<140 20 (13.6%) 40 (13.7%) 60 (13.7%) 40-<55  80 (54.4%) 145 (49.7%)225 (51.3%) ≥55 47 (32.0%) 107 (36.6%) 154 (35.1%)

MS disease duration was similar in both treatment groups. Baseline MRIresults are summarized in Table 4. The baseline MRI characteristics weresimilar in placebo and rituximab groups.

TABLE 4 Baseline MRI Results: Intent-to-Treat Subjects. MRI PlaceboRituximab All Subjects Endpoint (N = 147) (N = 292) (N = 439) Total 2gadolinium- enhancing lesion count N 147 290 437 Mean (SD) 0.5 (1.26)0.7 (2.96) 0.7 (2.52) Median 0.0 0.0 0.0 Minimum to 0-8 0-32 0-32maximum 0 110 (74.8%) 220 (75.9%) 330 (75.5%) 1 23 (15.6%) 44 (15.2%) 67(15.3%) 2 5 (3.4%) 10 (3.4%) 15 (3.4%) 3 5 (3.4%) 5 (1.7%) 10 (2.3%) ≥44 (2.7%) 11 (3.8%) 15 (3.4%) Total gadolinium- enhancing lesion volume(mm³) N 147 290 437 Mean (SD) 27.56 (81.86) 49.50 (220.32) 42.12(185.82) Median 0.00 0.00 0.00 Minimum to   0.00-556.40   0.00-2660.00  0.00-2660.00 maximum T2 lesion volume (mm³) N 147 290 437 Mean (SD)8850.86 9336.66 9173.25 (11808.95) (13744.94) (13113.98) Median 5199.505240.50 5220.70 Minimum to   73.83-74534.0  174.00-155303.0  73.83-155303.0 maximum Brain volume (cc) N 130 237 367 Mean (SD)1210.91 (128.89) 1202.92 (120.23) 1205.75 (123.25) Median 1209.5 1204.01207.0 Minimum to  642.0-1522.0 712.60-1508.0  642.0-1522.0 maximum MRI= magnetic resonance imaging.

Randomization was stratified according to study site; baseline diseaseseverity defined by EDSS (≤4.0, >4.0). Baseline EDSS is summarized inTable 5. As a result of the dynamic randomization, the percentage ofsubjects in each treatment group was similar at all levels of thestratification factors.

TABLE 5 Baseline Stratification Factors, Disease SeverityIntent-to-Treat Subjects. Stratification Placebo Rituximab All SubjectsFactor (n = 147) (n = 292) (n = 439) EDSS N 147 292 439 2 6 (4.1%) 8(2.7%) 14 (3.2%) 2.5 5 (3.4%) 11 (3.8%) 16 (3.6%) 3 11 (7.5%) 22 (7.5%)33 (7.5%) 3.5 21 (14.3%) 36 (12.3%) 57 (13.0%) 4 25 (17.0%) 47 (16.1%)72 (16.4%) 4.5 8 (5.4%) 19 (6.5%) 27 (6.2%) 5 6 (4.1%) 10 (3.4%) 16(3.6%) 5.5 10 (6.8%) 10 (3.4%) 20 (4.6%) 6 28 (19.0%) 81 (27.7%) 109(24.8%) 6.5 27 (18.4%) 48 (16.4%) 75 (17.1%) Mean (SD) 4.73 (1.395) 4.84(1.369) 4.80 (1.377) Median 4.50 5.00 5.00 Minimum to 2.0-6.5 2.0-6.52.0-6.5 maximum EDSS = Expanded Disability Status Scale.

Additionally, the two treatment groups were similar in other baselinedisease severity measures: EDSS, Kurtzke Functional System Scores, theMultiple Sclerosis Functional Composite Scale (MSFCS) score and theMSFCS components (Timed 25-Foot Walk, 9-Hole Peg Test, and PASAT-3).

Efficacy Results

The primary efficacy analysis for this trial compared the time toconfirmed disease progression, during the 96 week treatment period,between rituximab and placebo. Disease progression is defined as anincrease of ≥1.0 point from baseline EDSS (Kurtzke I Neurology33(11):1444-52 (1983)), if the baseline EDSS is between 2.0 and 5.5points (inclusive), or an increase of ≥0.5 point if the baseline EDSSis >5.5 points, for which change is not attributable to another etiology(e.g., fever, concurrent illness, MS relapse or exacerbation, orconcomitant medication).

Stratified analysis showed that rituximab did not significantly delaythe confirmed disease progression compared with placebo (p=0.1442,stratified log-rank). The percentage of patients progressing by 96 weekswas estimated to be 38.5% and 30.2% for the placebo and rituximabgroups, respectively (Table 6). Kaplan-Meier plots for the time toconfirmed disease progression are shown in the FIG. 8.

TABLE 6 The Time to Confirmed Disease Progression during TreatmentPeriod Intent-to-Treat Subjects. Placebo Rituximab (n = 147) (n = 292)No. of subjects who had CDP (%) 53 (36.1%) 83 (28.4%) No. of subjectswho censored (%) 94 (63.9%) 209 (71.6%) Stratified p-value Log-rank test0.1442 Stratified hazard ratio 0.773 (rel to placebo) 95% CI for(0.546-1.093) Proportion of subjects with CDP at week 24  6.9%  9.1% atweek 48 19.3% 20.2% at week 72 30.3% 28.0% at week 96 38.5% 30.2%

Secondary efficacy endpoints included change from baseline to Week 96 inthe total volume of T2 lesions and change from baseline to Week 96 inthe brain volume. A Hochberg-Bonferroni procedure was used to controlthe type I error rate in testing these two secondary endpoints. Thechange from baseline to Week 96 in the brain volume was notsignificantly different in the two treatment groups (p=0.6237). SeeTable 7.

TABLE 7 Change in Brain Volume from Baseline to Week 96. PlaceboRituximab (N = 130) (N = 237) P-value Volume at baseline (cm3) mean (SD)1211 (129) 1203 (120) median 1209.5 1204.0 Volume change from baselineto week 96 (LOCF) mean (SD) −9.9 (37.0) −10.8 (40.3) 0.62 median −14.0−13.1

A significant difference was observed between the two treatments for thechange in T2 lesion volume from baseline to week 96 (p=0.0008). Themedian increase in volume of the T2 lesion was 809.50 mm³ and 301.95 mm³in the placebo and rituximab groups, respectively. (See Table 8 and FIG.9).

Change from Baseline to Week 96 in the Total Volume of T2 Lesions onBrain MRI Scans Intent-to-Treat Subjects Placebo Rituximab (n = 147) (n= 292) p-Value Total volume of T2 lesions on brain MRI scan (mm3)Baseline N 147 290 Mean (SD) 8850.86 (11808.95)  9336.66 (13744.94)Median 5199.50 5240.50 Range   73.83-74534.00  174.00-155303.0 SE 973.99807.13 95% CI  (6925.93-10775.79)  (7748.06-10925.26) Week 96 N 147 290Mean (SD) 11055.55 (14536.29) 10843.80 (15827.44) Median 5526.60 5569.35Range   94.92-86232.00  179.30-170464.0 SE 1198.93 929.42 95% CI (8686.04-13425.05)  (9014.51-12673.09) Change from baseline to week 96N 147 290 Mean (SD) 2204.69 (4306.24) 1507.14 (3739.45) Median 809.50301.95 Range −8557.00-26367.00 −4031.00-24076.00 SE 355.17 219.59 95% CI(1502.74-2906.63) (1074.94-1939.33) Treatment −718.24 difference in LSmeans (vs. placebo) 95% CI of the (−1504.48, 68.00) difference in LSmeans ANOVA t-test 0.0733 (stratified) Friedman ranked 0.0008 ANOVA testANOVA t-test 0.0006 (stratified) on percent change from baseline to week96 Friedman ranked 0.0005 ANOVA test on percent change from baseline toweek 96

Analyses of all exploratory endpoints except the change in T2 lesionvolume, enlarging T2 lesion and new T2 lesion showed statisticallynon-significant differences between placebo and rituximab arms. Comparedto placebo, the rituximab group experienced significantly less increasein T2 lesion volume at Week 48 and 122 (p=0.0051 and 0.0222,respectively); had less new T2 lesion at week 48 and 96 (p<0.001); hadless enlarging T2 lesion count at week 48 and 96 (p=0.008 and 0.072,respectively).

Subgroup Analysis

Subgroup analysis for the primary endpoints included time to confirmeddisease progression according to the following demographic and baselinedisease characteristics: sites, age, gender, race, prior MS therapies,baseline EDSS, duration since MS symptom onset and baseline gadolinium(Gd) lesion, and baseline Multiple Sclerosis Severity Score (MSSS) (anindex of how fast the patient progressed; see Roxburgh et al. Neurology64; 1144-1151 (2005)).

The subgroup analysis results suggest a potential treatment effect inpatients who are younger, progressed more rapidly (higher MSSS) or withGd lesions at baseline (FIG. 10). Moreover, additive predictive effectsof age, Gd lesion at baseline and MSSS for the treatment effect havebeen verified using multivariate analysis method (FIG. 11 and FIG. 12).See also Table 9. Based on these findings with MSSS, a subgroup of thestudy population excluding older patients with longstanding disease andslow progression was selected using modified inclusion/exclusioncriteria (age ≤55, 3≤baseline EDSS≤6.5, excluding patients with diseaseduration >10 if their baseline EDSS<5 or disease duration >15 if theirbaseline EDSS≥5). A significant treatment effect was also shown for thissubgroup (stratified log-rank test P-value=0.01; FIG. 13).

TABLE 9 Time to Confirmed Disease Progression Subgroup Result Summary.Total CDP@wk 96 CDP@wk 96 P-value Subgroups N Placebo Rituximab HR HR95% CI (Log-rank) All patients 439 38.5% 30.2% n/a n/a 0.1442 (Primaryanalysis) Age <51 215 44.9% 27.5% 0.52 (0.32, 0.86) 0.0101 Gd+ 107 52.8%27.4% 0.41 (0.21, 0.80) 0.0069 Gd+ and age <51  72 51.6% 24.6% 0.33(0.14, 0.79) 0.0088 Gd+ and age <55  93 49.5% 29.1% 0.40 (0.19, 0.84)0.0126 MSSS >= 5 and 251 46.8% 29.6% 0.59 (0.38, 0.91)  .0163 age <55

Subgroup analyses suggest that PPMS patients with evidence of activedisease show significant clinical signs of treatment related benefit asmeasured by time to confirmed disease progression, as well as changefrom baseline in EDSS, MSFC, and T2 lesions on brain MRI (data notshown). Independent factors that appeared prognostic of diseaseprogression in the placebo group and potentially predictive of treatmentresponse in the rituximab group included the following: younger age,particularly age less than 51; presence of contrast enhancing lesions atbaseline on brain MRI; and higher MS severity score. These observationsserve a hypothesis generating role supporting a potential therapeuticbenefit of B-cell depletion on confirmed disease progression inappropriately selected progressive onset MS patients.

While this study failed to demonstrate primary efficacy in the overallPPMS population, subgroup efficacy analyses indicated that patients withcontrast enhancing brain MRI lesions at baseline potentially respondedto treatment with rituximab, with a 57% relative reduction in the hazard(1-HR) of confirmed disease progression in the treated group versusplacebo, which is largely but not entirely driven by a very high placeboConfirmed Disease Progression (CDP) rate of 52.8% at 96 weeks (FIG. 10).PPMS patients aged less than 51 may also have benefited, with a 43%relative reduction in the hazard of confirmed disease progression and aplacebo progression rate of 44.9%. While the presence of contrastenhancing lesions and age <51 were correlated, a post-hoc analysis ofthe 72 patients exhibiting both characteristics revealed a morepronounced apparent effect, with a 77% relative reduction in the hazardof confirmed disease progression (Table 9). In this subgroup the placeboprogression rate of 51.6% was not higher than the rate for all patientswith enhancing MRI lesions at baseline, but a lower rate of progressionin the rituximab group (24.6%) accounts for the potentially greater riskreduction with treatment. These OLYMPUS placebo data corroborate naturalhistory observations on the clinical and MRI heterogeneity of PPMSpatients (Sastre-Garriga et al. Neurology 65(4):633-5 (2005), Ingle etal. Brain 126(Pt 11):2528-36 (2003), Tremlett et al. Mult Scler.14(3):314-24 (2008), Tremlett et al. Neurology 65(12):1919-23 (2005),Kremenchutzky et al. Brain 129(Pt 3):584-94. (2006)). Furthermore, theMAGNIMS clinical and MRI cohort study described a subset of PPMSpatients with more inflammatory MRI activity early in the disease courseand a worse prognosis for disability progression (Ingle et al. J. NeurolNeurosurg Psychiatry 76(9):1255-8 (2005)); the OLYMPUS placebo dataappear to confirm these observations for the first time.

Example 3: A Phase III Study of Ocrelizumab in Progressive MultipleSclerosis

A Phase III, randomized, double-blind, parallel-group, multicenter studyto evaluate the safety and efficacy of 600 mg of ocrelizumab as comparedwith placebo in adults with progressive MS is performed.

A total of 630 progressive MS patients (315 with progressive onset and315 with relapsing onset MS) are enrolled and assigned (2:1randomization) to either ocrelizumab arm or placebo arm stratified bysite and type of multiple sclerosis. This study consists of thefollowing three periods that apply to all patients: a screening period,a treatment period and a treatment free follow up period. In the firstcourse of study drug treatment (300 mg ocrelizumab or placeboinfusion×2) are administered on Days 1 and 15. In subsequent treatmentcourses, patients are dosed (600 mg ocrelizumab single infusion) every24 weeks until the last patient enrolled receives his/her last course oftreatment to be administered at Week 96.

Prior to each study drug infusion, patients receive treatment with ananalgesic/antipyretic such as acetaminophen/paracetamol (1 grams) and ani.v. or oral antihistamine (such as diphenhydramine 50 mg), and 100 mgmethylprednisolone intravenously, or equivalents, to reduce theincidence of potential infusion reactions. In patients with CommonTerminology for Adverse Events (CTCAE) Grade 3 or higher (severe)infusion reactions with associated respiratory symptoms (stridor, wheezeor bronchospasm), additional treatment with bronchodilators may beindicated.

Routine laboratory studies are obtained throughout the study, withadditional tests following courses of study drug treatment. Immunepanel, serum human anti-human antibody (HAHA), and thyroid tests arealso conducted. Serum samples of all patients are collected forpharmacokinetic analysis and blood samples are collected for B-cellcount determination. B-cell counts are followed as a pharmacodynamicmarker of ocrelizumab.

Patient Population and Selection Criteria

The target population for this study includes patients with progressiveMS with or without a history of superimposed relapses. Patients withprogressive MS eligible for this study are characterized by a diagnosisin accordance with the revised McDonald criteria (2005) and a period of6 months or greater of documented irreversible loss of neurologicalfunction in the absence of relapses. Patients are selected with evidenceof active disease and higher risk for more rapid disability progression,using criteria identified as potential risk factors in previous clinicaltrials with progressive MS patients. These factors include younger age,evidence of inflammation in the cerebrospinal fluid (CSF) (oligoclonalbands or elevated IgG index), contrast enhancing lesions on brain MRI,high relapse activity superimposed on non-relapse related progression,and more rapid historical accumulation of disability.

All patients volunteering and eligible for participation in the studyare screened for conformance with the following inclusion and exclusioncriteria:

Inclusion Criteria include:

1. Diagnosis of Multiple Sclerosis in accordance with the revisedMcDonald criteria (2005).2. Progressive MS, characterized by documented, irreversible loss ofneurological function persisting for ≥6 months that cannot be attributedto clinical relapse.3. Ages 18-55 years inclusive.4. EDSS at screening from 3.0 to 6.5 points.5. Score of ≥2.0 on the Functional Systems (FS) scale for the pyramidalsystem or gait that is due to lower extremity findings.6. Presence of at least one of the following laboratory findings in aCSF specimen obtained during the screening period or documented withinthe previous 6 months as indicated by, for example, elevated IgG indexand/or IgG oligoclonal bands detected by isoelectric focusing.7. Presence of at least one of the following criteria:

-   -   Age <50    -   Gd+ lesions on brain MRI at screening or within 6 months of        screening    -   At least 1.5 point increase in EDSS over past 2 yrs not        attributable to relapse    -   Two relapses in past two years

Exclusion criteria include:

1. Relapsing remitting multiple sclerosis at screening (Visit 1)2. Disease duration from the onset of MS symptoms: more than 15 years inpatients with an EDSS at screening >5.0 or more than 10 years inpatients with an EDSS at screening <5.0.

Efficacy Analysis

The primary efficacy endpoint is the time to confirmed diseaseprogression. Disease progression is defined as an increase of ≥1.0 pointfrom baseline EDSS, if the baseline EDSS is between 2.0 and 5.5 points(inclusive), or an increase of ≥0.5 points, if the baseline EDSS is >5.5points, for which change is not attributable to another etiology (e.g.,fever, concurrent illness, MS relapse or exacerbation, or concomitantmedication).

The EDSS is based on a standard neurological examination; the sevencategories of the EDSS representing functional systems (pyramidal,cerebellar, brainstem, sensory, bowel and bladder, visual, and cerebraland/or mental, plus “other”) are rated and scored (collectively,functional system scores or FSS). Each score of the FSS is an ordinalclinical rating scale ranging from 0 to 5 or 6. These ratings are thenused in conjunction with observations and information concerningambulation and use of assistive devices to determine the EDSS score. TheEDSS is a disability scale that ranges in 0.5-point steps from 0(normal) to 10 (death).

The secondary efficacy endpoints in support of the primary efficacyendpoint include: change from baseline to Week 120 in the total volumeof T2 lesions on brain MRI scan, change from baseline to Week 120 in the25-foot timed walk, time to confirmed disease progression, withconfirmation occurring at least 24 weeks (≥168 days) after initialdisease progression.

Assessment of Relapse

Patients are evaluated for relapses by the treating investigator at eachvisit throughout the study and, if necessary, at unscheduled visits toconfirm relapses occurring between the visits. To meet the criteria fora protocol-defined relapse, the relapse is defined as the occurrence ofnew or worsening neurological symptoms attributable to MS andimmediately preceded by a relatively stable or improving neurologicalstate of least 30 days. Symptoms must persist for >24 hours and shouldnot be attributable to confounding clinical factors (e.g., fever,infection, injury, adverse reactions to concomitant medications). Thenew or worsening neurological symptoms must be accompanied by objectiveneurological worsening consistent with an increase of at least half astep on the EDSS, or 2 points on one of the appropriate FSS, or 1 pointon two or more of the appropriate FSS. The change must affect theselected FSS (i.e., pyramidal, ambulation, cerebellar, brainstem,sensory, or visual). Episodic spasms, sexual dysfunction, fatigue, moodchange or bladder or bowel urgency or incontinence do not suffice toestablish a relapse.

Brain MRI Imaging

Magnetic resonance imaging of the brain and cervical spinal cord areobtained at multiple time points during this study, including atbaseline. The brain MRI includes the acquisition of the following scansat each time point: T2-weighted MRI scan and T1-weighted MRI scan(without gadolinium-enhancement).

Change from baseline to Week 120 in the total volume of T2 lesions andtimed 25 foot walk are compared between ocrelizumab and placebo usingranked analysis of variance. The model includes the two stratificationfactors noted in the primary analysis.

Example 4: A Phase III Study of Ocrelizumab in Primary ProgressiveMultiple Sclerosis

A Phase III, randomized, double-blind, parallel-group, multicenter studyto evaluate the safety and efficacy of one of two dose regimens ofocrelizumab as compared with placebo in adults with primary progressiveMS is performed.

The two ocrelizumab dose regimens under investigation are as follows: 1)ocrelizumab 1000 mg dose regimen: consisting of a dual infusion of 1000mg for the first treatment cycle followed by single infusions of 1000 mgfor the subsequent treatment cycles and 2) ocrelizumab 600 mg doseregimen: consisting of a dual infusion of 300 mg for the first treatmentcycle followed by single infusions of 600 mg for the subsequenttreatment cycles.

A total of 630 primary progressive MS patients are enrolled and assigned(2:1 randomization) to either ocrelizumab arm or placebo arm stratifiedby site and type of multiple sclerosis. This study consists of thefollowing three periods that apply to all patients: a screening period,a treatment period and a safety follow up period. In the first course ofstudy drug treatment (ocrelizumab or placebo infusion x 2) areadministered on Days 1 and 15. In subsequent treatment courses, patientsare dosed (ocrelizumab or placebo single infusion) every 24 weeks untilthe last patient enrolled receives his/her last course of treatment tobe administered at Week 96.

Prior to each study drug infusion, patients receive treatment with ananalgesic/antipyretic such as acetaminophen/paracetamol (1 grams) and ani.v. or oral antihistamine (such as diphenhydramine 50 mg), and 100 mgmethylprednisolone intravenously, or equivalents, to reduce theincidence of potential infusion reactions. In patients with CommonTerminology for Adverse Events (CTCAE) Grade 3 or higher (severe)infusion reactions with associated respiratory symptoms (stridor, wheezeor bronchospasm), additional treatment with bronchodilators may beindicated.

Routine laboratory studies are obtained throughout the study, withadditional tests following courses of study drug treatment. Immunepanel, serum human anti-human antibody (HAHA), and thyroid tests arealso conducted. Serum samples of all patients are collected forpharmacokinetic analysis and blood samples are collected for B-cellcount determination. B-cell counts are followed as a pharmacodynamicmarker of ocrelizumab.

Patient Population and Selection Criteria

The target population for this study includes patients with primaryprogressive MS. Patients with primary progressive MS eligible for thisstudy are characterized by a diagnosis in accordance with the revisedMcDonald criteria (2005). Patients are selected with evidence of activedisease and higher risk for more rapid disability progression, usingcriteria identified as potential risk factors in previous clinicaltrials with progressive MS patients. These factors include younger age,evidence of inflammation in the cerebrospinal fluid (CSF) (oligoclonalbands or elevated IgG index), and more rapid historical accumulation ofdisability.

All patients volunteering and eligible for participation in the studyare screened for conformance with the following inclusion and exclusioncriteria:

Inclusion Criteria include:

1. Diagnosis of Primary Progressive Multiple Sclerosis in accordancewith the revised McDonald criteria (2005).2. Ages 18-55 years inclusive.3. EDSS at screening from 3.0 to 6.5 points.4. Score of ≥2.0 on the Functional Systems (FS) scale for the pyramidalsystem that is due to lower extremity findings.5. Documented history or presence at screening of at least one of thefollowing laboratory findings in a CSF specimen as indicated by,elevated IgG index and/or IgG oligoclonal bands detected by isoelectricfocusing.6. Disease duration from the onset of MS symptoms: less than 15 years inpatients with an EDSS at screening >5.0 or less than 10 years inpatients with an EDSS at screening ≤5.0.

Exclusion criteria include:

1. History of relapsing remitting, secondary progressive, or progressiverelapsing multiple sclerosis at screening (Visit 1).

Efficacy Analysis

The primary efficacy endpoint is the time to confirmed diseaseprogression. Disease progression is defined as an increase of ≥1.0 pointfrom baseline EDSS, if the baseline EDSS is between 2.0 and 5.5 points(inclusive), or an increase of ≥0.5 points, if the baseline EDSS is >5.5points, for which change is not attributable to another etiology (e.g.,fever, concurrent illness, MS relapse or exacerbation, or concomitantmedication).

The EDSS is based on a standard neurological examination; the sevencategories of the EDSS representing functional systems (pyramidal,cerebellar, brainstem, sensory, bowel and bladder, visual, and cerebraland/or mental, plus “other”) are rated and scored (collectively,functional system scores or FSS). Each score of the FSS is an ordinalclinical rating scale ranging from 0 to 5 or 6. These ratings are thenused in conjunction with observations and information concerningambulation and use of assistive devices to determine the EDSS score. TheEDSS is a disability scale that ranges in 0.5-point steps from 0(normal) to 10 (death).

The secondary efficacy endpoints in support of the primary efficacyendpoint include: change from baseline to Week 120 in the total volumeof T2 lesions on brain MRI scan, change from baseline to Week 120 in the25-foot timed walk, time to confirmed disease progression, withconfirmation occurring at least 24 weeks (≥168 days) after initialdisease progression.

Assessment of Relapse

Patients are evaluated for relapses by the treating investigator at eachvisit throughout the study and, if necessary, at unscheduled visits toconfirm relapses occurring between the visits. To meet the criteria fora protocol-defined relapse, the relapse is defined as the occurrence ofnew or worsening neurological symptoms attributable to MS andimmediately preceded by a relatively stable or improving neurologicalstate of least 30 days. Symptoms must persist for >24 hours and shouldnot be attributable to confounding clinical factors (e.g., fever,infection, injury, adverse reactions to concomitant medications). Thenew or worsening neurological symptoms must be accompanied by objectiveneurological worsening consistent with an increase of at least half astep on the EDSS, or 2 points on one of the appropriate FSS, or 1 pointon two or more of the appropriate FSS. The change must affect theselected FSS (i.e., pyramidal, ambulation, cerebellar, brainstem,sensory, or visual). Episodic spasms, sexual dysfunction, fatigue, moodchange or bladder or bowel urgency or incontinence do not suffice toestablish a relapse.

Brain MRI Imaging

Magnetic resonance imaging of the brain and cervical spinal cord areobtained at multiple time points during this study, including atbaseline. The brain MRI includes the acquisition of the following scansat each time point: T2-weighted MRI scan and T1-weighted MRI scan(without gadolinium-enhancement).

Change from baseline to Week 120 in the total volume of T2 lesions andtimed 25 foot walk are compared between ocrelizumab and placebo usingranked analysis of variance. The model includes the two stratificationfactors noted in the primary analysis.

1. A method of treating progressive multiple sclerosis in a patientcomprising administering to the patient an effective amount of ananti-CD20 antibody, wherein treatment is based upon the patient havingone or more characteristics selected from the group consisting of (a) anage less than about 55 years, (b) one or more gadolinium staininglesions, (c) at least about a one point increase in Expanded DisabilityStatus Scale (EDSS) over two years prior to starting treatment, and (d)a Multiple Sclerosis Severity Score (MSSS) greater than about 5 points.2-38. (canceled)
 39. A method of treating multiple sclerosis in apatient comprising administering an effective amount of ocrelizumab tothe patient to provide an initial ocrelizumab exposure of between about0.3 to about 0.6 grams followed by a second ocrelizumab exposure ofbetween about 0.3 to about 0.6 grams, the second exposure not beingprovided until from about 16 to 60 weeks from the initial exposure, andeach of the ocrelizumab exposures is provided to the patient as one ortwo doses of ocrelizumab. 40-46. (canceled)
 47. An article ofmanufacture comprising: (a) a container comprising ocrelizumab; and (b)a package insert with instructions for treating multiple sclerosis in apatient, wherein the instructions indicate that an amount of ocrelizumabis administered to the patient that is effective to provide an initialocrelizumab exposure of between about 0.3 to about 0.6 grams followed bya second ocrelizumab exposure of between about 0.3 to about 0.6 grams,the second exposure not being administered until from about 16 to 60weeks from the initial exposure, and each of the ocrelizumab exposuresis provided to the patient as one or two doses of ocrelizumab.